Carbohydrate Moieties as Vaccine Candidates: meeting summary.

In September 2007, a meeting entitled 'Carbohydrate Moieties as Vaccine Candidates' was held at the National Institutes of Health (Bethesda, MD). This meeting brought together scientists from a number of disciplines to address issues concerning carbohydrate moieties as targets for vaccines for a variety of pathogens and tumors. In addition, the meeting participants addressed fundamental topics of glycoimmunology including the recognition of glycotopes by B and T lymphocytes, the ontogeny of anti-carbohydrate immune responses, peptide mimicry, carbohydrate antigen processing pathways and adjuvants. One session reported progress in the development of new tools such as computational algorithms, glycan arrays and oligosaccharide synthesis and their application to carbohydrate vaccine research. The session titles were: (1) immune response to bacterial carbohydrate antigens; (2) immune response to glycolipids; (3) immune response to carbohydrate antigens on other microbes and on tumors; (4) novel vaccine approaches; (5) novel tools in carbohydrate vaccine research; (6) bench to bedside: carbohydrate moieties as vaccine immunopotentiators.

Biofilm Formation by Neisseria gonorrhoeae.

Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could form a biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10 microM sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain 1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with water channels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growth conditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmed that organisms were embedded in a continuous matrix with membranous structures spanning the biofilm. These studies also demonstrated that N. gonorrhoeae has the capability to form a matrix in the presence and absence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and the lectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in the biofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac and sialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primary urethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Our studies demonstrated that N. gonorrhoeae can form biofilms in continuous-flow chambers and on living cells. Studies of these biofilms may have implications for understanding asymptomatic gonococcal infection.

Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans.

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with alpha2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

Nontypeable Haemophilus influenzae in the lower respiratory tract of patients with chronic bronchitis.

The frequency of colonization and intracellular localization of nontypeable Haemophilus influenzae (NTHi) in the lower respiratory tract was determined in healthy adults and in clinically stable and acutely ill chronic bronchitis (CB) patients. NTHi was recovered from bronchial wash or bronchial brush specimens in 6 of 23 (26%) stable CB patients and in 1 of 15 (7%) CB patients with a respiratory exacerbation. No NTHi (0 of 26) was recovered from lower tract specimens of healthy adults undergoing anesthesia for elective surgery. Molecular typing of NTHi strains revealed that five of nine patients with stable CB had different strains in upper respiratory tract and bronchial wash/brush specimens collected simultaneously. Four stable patients with CB had different strains recovered on repeat bronchoscopy. These results demonstrate the frequent colonization of the lower airways of stable CB patients with multiple strains of NTHi. Bronchial biopsies also were examined for intracellular NTHi by in situ hybridization and immunofluorescence microscopy. Intracellular NTHi were found in 0 of 7 healthy adults, 8 of 24 patients with clinically stable CB, and 13 of 15 acutely ill CB patients. This observation suggests a role for intracellular infection by NTHi in the pathogenesis of exacerbations of CB.

Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor.

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP-R) and the terminal lactosamine of lacto-N-neotetraose-expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP-R traffics to the cell surface after gonococcal challenge. Co-localization of ASGP-R with gonococci was observed. As ASGP-R-mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co-localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin-coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(beta1-4)GlcNac(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP-R ligand, significantly reduced invasion. A dose-response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH-deacylated 1291 LOS. These studies indicated that an interaction between lacto-N-neotetraose-terminal LOS and ASGP-R allows gonococcal entry into PHUECs.

The role of complement receptor 3 (CR3) in Neisseria gonorrhoeae infection of human cervical epithelia.

Neisseria gonorrhoeae is an important sexually transmitted pathogen and a major cofactor in HIV-1 infection. This organism uses different mechanisms to infect male and female genital tract epithelia. Receptor-mediated endocytosis of N. gonorrhoeae is the principle mechanism of entry into male urethral epithelial cells. Infection in men leads to a pronounced inflammatory response. In contrast, N. gonorrhoeae infection in women induces ruffling of the cervical epithelia, allowing a macropinocytic mechanism of entry. Infection in women is frequently asymptomatic, suggesting suppression of the inflammatory response. N. gonorrhoeae-induced membrane ruffling and inflammation suppression are consistent with the ability of this bacterium to enter cervical epithelial cells, in vitro and in vivo, by interaction with complement receptor 3 (CR3), a receptor that does not trigger an inflammatory response. This receptor is present on cervical epithelial cells but not on male urogenital tract epithelia. N. gonorrhoeae engagement of CR3 initiates a unique mechanism of bacterial-induced membrane ruffling and internalization. These studies explain why the pathology of N. gonorrhoeae infection differs between males and females. Additionally, the observation that this receptor is present on cervical epithelia may provide insight into the pathogenesis of other sexually transmitted pathogens.

Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling.

Non-typeable Haemophilus influenzae (NTHi) invades host cells by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infection on host cell signalling and its role in NTHi invasion was examined. The infection of human bronchial epithelial cells with NTHi 2019 increased cytosolic Ca2+ levels, and the invasion of bronchial cells by NTHi 2019 was inhibited by pretreatment with the cell-permeant intracellular Ca2+ chelator BAPTA-AM (P = 0.022) or thapsigargin (P = 0.016). Cytosolic inositol phosphate (IP) levels were also increased after infection with NTHi 2019 (P < 0.001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019-stimulated IP production in a dose-dependent manner. NTHi 2019 invasion was inhibited by pertussis toxin (PTX) and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002. The less invasive strain NTHi 7502 also initiated IP production, but was unaffected by PAF receptor antagonist or PTX. These data demonstrate that the binding of the PAF receptor by NTHi initiates receptor coupling to a PTX-sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. Moreover, the data suggest that NTHi strains initiate cell signalling and invade by different mechanisms, and that invasion mediated by PAF receptor activation is more efficient than macropinocytosis.

Invasion of human fallopian tube epithelium by Escherichia coli expressing combinations of a gonococcal porin, opacity-associated protein, and chimeric lipo-oligosaccharide.

The transepithelial migration of Escherichia coli that expressed all possible combinations of a plasmid-encoded gonococcal porin (Por), opacity-associated protein (Opa), and 3F11 lipo-oligosaccharide (LOS) epitope was investigated. Surface expression of Por mediated selective changes in E. coli antibiotic susceptibility, and coexpression of Opa and the 3F11 LOS epitope mediated bacterial clumping (P<.01). In the human fallopian tube organ-culture model, Opa-producing variants attached up to 44-fold better than control bacteria (P<.01), and Por-producing variants exceeded submucosal invasion of control bacteria by 500-fold (P<.01). Opa and Por each facilitated intracellular invasion 20-40-fold (P<.01). In dual expresser variants, the 3F11 LOS epitope markedly reduced attachment and invasion mediated by Opa or Por. The LOS inhibitory effect was curbed when all 3 factors were expressed, which suggests an additional interaction of the 3 factors at the bacterial surface. Por, Opa, and LOS play important roles in Neisseria gonorrhoeae trafficking across human fallopian tube epithelium.

The mimicry of human glycolipids and glycosphingolipids by the lipooligosaccharides of pathogenic neisseria and haemophilus.

It has been known for many years that bacteria can induce autoimmune responses in humans resulting in serious disease. Recent work has shown that a number of bacteria that colonize human mucosal surfaces exclusively express antigens on their surfaces which are molecular mimics of glycosphingolipids found on human cells. These structures are important in the pathogenesis of Neisseria and Haemophilus species for both immune evasion and in the adherence and invasion of human cells. There is no evidence that colonization or infections by these bacterial species is associated with autoimmune disease.

Modulation of gonococcal piliation by regulatable transcription of pilE.

The gonococcal pilus, a member of the type IV family of pili, is composed of numerous monomers of the pilin protein and plays an important role in the initiation of disease by providing the primary attachment of the bacterial cell to human mucosal tissues. Piliation also correlates with efficient DNA transformation. To investigate the relationships between these pilus-related functions, the piliation state, and the availability of pilin, we constructed a derivative of MS11-C9 (DeltapilE1) in which the lacIOP regulatory sequences control pilE transcription. In this strain, MS11-C9.10, the steady-state levels of pilin mRNA and protein directly correlate with the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) in the growth medium and can reach near-wild-type levels of expression. Transmission electron microscopy (TEM) demonstrated that the number of pili per cell correlated with the steady-state expression levels: at a low level of transcription, single long pili were observed; at a moderate expression level, many singular and bundled pili were expressed; and upon full gene expression, increased lateral association between pili was observed. Analysis of pilus assembly by TEM and epithelial cell adherence over a time course of induction demonstrated that pili were expressed as early as 1 h postinduction. Analysis at different steady-state levels of transcription demonstrated that DNA transformation efficiency and adherence of MS11-C9.10 to transformed and primary epithelial cells also correlated with the level of piliation. These data show that modulation of the level of pilE transcription, without a change in pilE sequence, can alter the number of pili expressed per cell, pilus bundling, DNA transformation competence, and epithelial cell adherence of the gonococcus.

Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions.

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.

Isolation and analysis of radiolabeled meningococcal endotoxin.

The Gram-negative pathogen Neisseria meningitidis, is one of the leading causes of bacterial meningitis worldwide (1). The host range for this organism is restricted to humans, where it colonizes the mucosal epithelium of the upper airway. It occasionally disseminates causing invasive disease (sepsis, disseminated intravascular coagulation [DIC], meningitis). Epidemic meningococcal meningitis is a major health problem, most notably in sub-Saharan Africa. In 1999, an outbreak of meningococcal disease spread across Guinea-Bissau, a region that is part of what is commonly called the African meningitis belt (2). There were 2,169 reported cases and 404 deaths resulting from meningococcal disease in this outbreak from Jan. 1 to April 5, 1999. Also in 1999, there were reported outbreaks in Sudan (22,000 cases and 1,600 deaths) Rwanda (29 cases and 11 deaths), Angola (253 cases and 147 deaths), Ethiopia (126 cases and 4 deaths) and Senegal (2,709 cases and 372 deaths) (2). According to the World Health Organization (WHO), each year approx 500,000 cases of meningitis and 50,000 deaths are attributable to N. meningitidis worldwide. In the United States, meningococcal disease is less common, although small outbreaks are reported each year (3).

Vibrio fischeri lipopolysaccharide induces developmental apoptosis, but not complete morphogenesis, of the Euprymna scolopes symbiotic light organ.

During initiation of the association between the squid host Euprymna scolopes and its bacterial partner Vibrio fischeri, the bacteria induce dramatic morphogenesis of the host symbiotic organ, a portion of which involves the signaling of widespread apoptosis of the cells in a superficial ciliated epithelium on the colonized organ. In this study, we investigated the role in this process of lipopolysaccharide (LPS), a bacterial cell-surface molecule implicated in the induction of animal cell apoptosis in other systems. Purified V. fischeri LPS, as well as the LPS of V. cholerae, Haemophilus influenzae, Escherichia coli, and Shigella flexneri, added in the concentration range of pg/ml to ng/ml, induced apoptosis in epithelial cells 10- to 100-fold above background levels. The absence of species specificity suggested that the conserved lipid A portion of the LPS was the responsible component of the LPS molecule. Lipid A from V. fischeri, E. coli, or S. flexneri induced apoptosis. In addition, strains of H. influenzae carrying a mutation in the htrB gene, which is involved in the synthesis of virulent lipid A, showed a diminished ability to induce apoptosis of host cells. Confocal microscopy using fluorescently labeled LPS indicated that the LPS behaves similar to intact bacterial symbionts, interacting with host cells in the internal crypt spaces and not directly with the superficial epithelium. Although LPS was able to induce apoptosis, it did not induce the full morphogenesis of the ciliated surface, suggesting that multiple signals are necessary to mediate the development of this animal-bacterial mutualism.

Lipooligosaccharide P(k) (Galalpha1-4Galbeta1-4Glc) epitope of moraxella catarrhalis is a factor in resistance to bactericidal activity mediated by normal human serum.

Moraxella catarrhalis is a respiratory pathogen responsible for acute bacterial otitis media in children and exacerbation of chronic bronchitis in adults. M. catarrhalis strains are frequently resistant to the bactericidal activity of normal human serum. In order to determine if the lipooligosaccharide (LOS) of M. catarrhalis has a role in serum resistance, the UDP-glucose-4-epimerase (galE) gene was identified, cloned, and sequenced and a deletion/insertion mutation was introduced into M. catarrhalis strain 2951. GalE enzymatic activity, measured in whole-cell lysates, was ablated in M. catarrhalis 2951 galE. Mass spectrometric analysis of LOS isolated with hot phenol-water confirmed that strain 2951 produced a type A LOS. These studies showed that the LOS from 2951 galE had lost two hexose residues due to the galE mutation and that the resultant LOS structure lacked the (Galalpha1-4Galbeta1-4Glc) P(k) epitope found on M. catarrhalis 2951. Wild-type M. catarrhalis 2951 is resistant to complement-mediated serum bactericidal activity. In contrast, a greater than 2-log(10)-unit reduction in CFU occurred after incubation of 2951 galE in either 50 or 25% pooled human serum (PNHS), and CFU in 10% PNHS decreased by about 1 log(10) unit. These studies suggest that the P(k) epitope of the LOS may be an important factor in the resistance of M. catarrhalis to the complement-mediated bactericidal effect of normal human serum.

Neisseria gonorrhoeae elicits membrane ruffling and cytoskeletal rearrangements upon infection of primary human endocervical and ectocervical cells.

Neisseria gonorrhoeae is a strict human pathogen that is, primarily, transmitted by close sexual contact with an infected individual. Gonococcal infection of the male urogenital tract has been well studied in experimental human models and in urethral cell culture systems. Recent studies, using tissue culture cell systems, have suggested a role for the cervical epithelium in gonococcal infection of females; however, the nature of gonococcal infection of the normal uterine cervix remains controversial. To address this enigma, we have developed two primary human cervical epithelial cell systems from surgical biopsies. Gonococcal infection studies and electron microscopy show that N. gonorrhoeae is capable of infecting and invading both the endo- and the ectocervix. Invasion was found to occur primarily in an actin-dependent manner, but it does not appear to require de novo protein synthesis by either the bacterium or the host cervical cell. Membrane ruffles appear to be induced in response to gonococci. Consistent with membrane ruffling, gonococci were found residing within macropinosomes, and a concentrated accumulation of actin-associated proteins was observed to occur in response to gonococcal infection. Electron microscopy of clinically derived cervical biopsies show that lamellipodia formation and cytoskeletal changes, suggestive of membrane ruffles, also occur in the cervical epithelium of women with naturally acquired gonococcal cervicitis. These studies demonstrate the ability of N. gonorrhoeae to infect and invade both the endo- and the ectocervix of the normal uterine cervix. Gonococcal induced ruffling is a novel finding and may be unique to the cervical epithelium.

Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor.

Adherence and invasion are thought to be key events in the pathogenesis of non-typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS-coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB:ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB:ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co-localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti-ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.

Interaction of the gonococcal porin P.IB with G- and F-actin.

The invasion of epithelial cells by N. gonorrheae is accompanied by formation of a halo of actin filaments around the enveloped bacterium. The transfer of the bacterial major outer membrane protein, porin, to the host cell membrane during invasion makes it a candidate for a facilitator for the formation of this halo. Western analysis shows here that gonococcal porin P.IB associates with the actin cytoskeleton in infected cells. Using the pyrene-labeled Mg forms of yeast and muscle actins, we demonstrate that under low ionic strength conditions, P.IB causes formation of filamentous actin assemblies, although they, unlike F-actin, cannot be internally cross-linked with N,N'-4-phenylenedimaleimide (PDM). In F-buffer, low porin concentrations appear to accelerate actin polymerization. Higher P.IB concentrations lead to the formation of highly decorated fragmented F-actin-like filaments in which the actin can be cross-linked by PDM. Co-assembly of P.IB with a pyrene-labeled mutant actin, S(265)C, prevents formation of a pyrene excimer present with labeled S(265)C F-actin alone. Addition of low concentrations of porin to preformed F-actin results in sparsely decorated F-actin. Higher P.IB concentrations extensively decorate the filaments, thereby altering their morphology to a state like that observed when the components are copolymerized. With preformed labeled S(265)C F-actin, P.IB quenches the pyrene excimer. This decrease is prevented by the F-actin stabilizers phalloidin and to a lesser extent beryllium fluoride. P.IB's association with the actin cytoskeleton and its ability to interact with and remodel actin filaments support a direct role for porin in altering the host cell cytoskeleton during invasion.

Gonococcal lipooligosaccharide is a ligand for the asialoglycoprotein receptor on human sperm.

In the present study, we show that Neisseria gonorrhoeae lipooligosaccharide (LOS) can bind to the asialoglycoprotein receptor (ASGP-R) on human sperm. This work demonstrates the presence of ASGP-R on human sperm. Binding of purified ASGP-R ligand decreased in the presence of gonococci. Binding of purified iodinated gonococcal LOS identified a protein of molecular weight corresponding to that of human ASGP-R. The presence of excess unlabelled LOS blocked binding of iodinated gonococcal LOS. Binding of wild-type gonococcal LOS to sperm was higher than that of mutant LOS lacking the galactose ligand for ASGP-R. These data suggest that the ASGP-R on human sperm cells recognizes and binds wild-type gonococcal LOS. This interaction may contribute to the transmission of gonorrhea from infected males to their sexual partners.

A pilus-deficient mutant of Haemophilus ducreyi is virulent in the human model of experimental infection.

Haemophilus ducreyi expresses fine tangled pili, which are composed predominantly of a major subunit (FtpA). Confocal microscopy showed that an FtpA-specific monoclonal antibody bound to bacteria in biopsy samples obtained from infected human volunteers. To test the role of pili in pathogenesis, an isogenic mutant (35000HP-SMS1) was constructed by insertionally inactivating ftpA. 35000HP-SMS1 did not express FtpA and was nonpiliated but was otherwise identical to its parent, 35000HP. Seven healthy adults were challenged on the upper arm with the isogenic isolates in a double-blinded, escalating dose-response study. Sites inoculated with the mutant produced papules and pustules at rates similar to the rates observed at sites inoculated with the parent. The recovery rate of H. ducreyi from cultures and the histopathology of biopsy samples obtained from pustules inoculated with 35000HP or 35000HP-SMS1 were similar. Although pili are expressed in vivo, FtpA is not required for pustule formation in the human challenge model.

Characterization of chimeric lipopolysaccharides from Escherichia coli strain JM109 transformed with lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae.

Previously, we reported the expression of chimeric lipopolysaccharides (LPS) in Escherichia coli strain JM109 (a K-12 strain) transformed with plasmids containing Haemophilus influenzae lipooligosaccharide synthesis genes (lsg) (Abu Kwaik, Y., McLaughlin, R. E., Apicella, M. A., and Spinola, S. M. (1991) Mol. Microbiol. 5, 2475-2480). In this current study, we have analyzed the O-deacylated LPS and free oligosaccharides from three transformants (designated pGEMLOS-4, pGEMLOS-5, and pGEMLOS-7) by matrix-assisted laser desorption ionization, electrospray ionization, and tandem mass spectrometry techniques, along with composition and linkage analyses. These data show that the chimeric LPS consist of the complete E. coli LPS core structure glycosylated on the 7-position of the non-reducing terminal branch heptose with oligosaccharides from H. influenzae. In pGEMLOS-7, the disaccharide Gal1--> 3GlcNAc1--> is added, and in pGEMLOS-5, the structure is extended to Gal1-->4GlcNAc1-->3Gal1-->3GlcNAc1-->. PGEMLOS-5 LPS reacts positively with monoclonal antibody 3F11, an antibody that recognizes the terminal disaccharide of lacto-N-neotetraose. In pGEMLOS-4 LPS, the 3F11 epitope is apparently blocked by glycosylation on the 6-position of the terminal Gal with either Gal or GlcNAc. The biosynthesis of these chimeric LPS was found to be dependent on a functional wecA (formerly rfe) gene in E. coli. By using this carbohydrate expression system, we have been able to examine the functions of the lsg genes independent of the effects of other endogenous Haemophilus genes and expressed proteins.