Biomedical subjects

M A Apicella

Publications and source records attributed to M A Apicella.

At least 37 records · Page 2Linked to original sources

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

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.

Actins↗

PubMed1999

Use of a bilayer stacking gel to improve resolution of lipopolysaccharides and lipooligosaccharides in polyacrylamide gels.

Lipopolysaccharide (LPS) and lipooligosaccharide (LOS) are important antigenic and integral components of the outer membrane of Gram-negative bacteria. Alteration or heterogeneity of LPS/LOS structure is most often assessed by alteration of electrophoretic band profiles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In order to discern minor differences in the electrophoretic profile of closely spaced bands, particularly the low molecular weight bands of LOS, optimum resolution is required. Unfortunately, many publications of LPS/LOS in polyacrylamide gels show a diffuse, smeared pattern without discernible bands. We report here a formulation for polyacrylamide gels that reproducibly yields LPS/LOS bands with sharp resolution. A key feature of this formulation is the use of a separate comb gel containing electrode buffer layered on top of the conventional stacking gel.

Acrylic Resins↗

PubMed1999

The R-type pyocin of Pseudomonas aeruginosa C is a bacteriophage tail-like particle that contains single-stranded DNA.

Pseudomonas aeruginosa R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from P. aeruginosa C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digested P. aeruginosa C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18 P. aeruginosa strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of P. aeruginosa C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonal prt genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from P. aeruginosa C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of P. aeruginosa C.

Amino Acid Sequence↗

PubMed1999

Relationship between UDP-glucose 4-epimerase activity and oligoglucose glycoforms in two strains of Neisseria meningitidis.

Sodium dodecyl sulfate-polyacrylamide gel analysis of lipooligosaccharide (LOS) from Neisseria meningitidis has demonstrated considerable microheterogeneity in the variable region of LOS due to the presence of novel glycoforms. As a step toward understanding the basis for the expression of these novel glycoforms, we have examined the LOS structures and UDP-glucose 4-epimerase (epimerase) activity levels in two strains (NMB and MA-1) and their respective galE mutants. Strain NMB was found to have low epimerase activity and to contain multiple glycoforms, some of which appear to contain only glucose sugars. The galE mutant had only the oligoglucose glycoforms. Strain MA-1 had higher epimerase activity at both log and stationary phases (2- and 12.5-fold, respectively) and one glycoform with a putative lactosyl structure. Strain MA-1 galE had two glycoforms that contained one or two glucose residues. To understand the molecular basis for the different epimerase activities, we examined the predicted amino acid sequences of the respective galE open reading frames and determined the relative amounts of GalE protein. We found no significant differences between the predicted amino acid sequence of the GalE protein in NMB and that in MA-1. We observed no significant differences in the level of GalE protein between MA-1 and NMB at exponential or stationary phase. We also observed an 8.2-fold drop in epimerase activity in NMB between the log and stationary phases that was not due to the GalE protein level or low glucose levels.

Amino Acid Sequence↗

PubMed1999

Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry.

Nontypeable Haemophilus influenzae is an exclusive human pathogen which infects the respiratory epithelium. We have initiated studies to explore the interaction of the nontypeable H. influenzae strain 2019 with primary human airway epithelial cells by electron and confocal microscopy. Primary human airway cell cultures were established as monolayers on glass collagen-coated coverslips or on semipermeable membranes at an air-fluid interface. Scanning electron microscopy indicated that bacteria adhered to nonciliated cells in the population. The surface of infected cells showed evidence of cytoskeletal rearrangements manifested by microvilli and lamellipodia extending toward and engaging bacteria. Confocal microscopic analysis demonstrated that infection induced actin polymerization with an increase in cortical actin as well as evidence of actin strands around the bacteria. Transmission electron microscopic analysis showed lamellipodia and microvilli surrounding organisms, as well as organisms adherent to the cell surface. These studies also demonstrated the presence of bacteria within vacuoles inside of airway cells. Confocal microscopic studies with Texas red-labeled dextran (molecular weight, 70,000) indicated that H. influenzae cells were entering cells by the process of macropinocytosis. These studies indicate that nontypeable H. influenzae can initiate cytoskeletal rearrangement within human airway epithelium, resulting in internalization of the bacteria within nonciliated human airway epithelial cells by the process of macropinocytosis.

Bacterial Adhesion↗

PubMed1998

Nonopsonic phagocytosis of group C Neisseria meningitidis by human neutrophils.

Although complement-mediated bactericidal activity in serum has long been known to be very important in host defense against Neisseria meningitidis, recent studies have shown that opsonic phagocytosis by neutrophils is also important. The purpose of this study was to determine if endemic group C N. meningitidis strains were susceptible to nonopsonic (complement- and antibody-independent) phagocytosis by human neutrophils, which is a well-described phenomenon for Neisseria gonorrhoeae. Gonococci that possess one or more of a group of heat-modifiable outer membrane proteins (called opacity-associated [Opa] proteins) are phagocytosed by neutrophils in the absence of serum. We found that four serogroup C meningococcal strains bearing the lacto-N-neotetraose (LNnT) structure on lipooligosaccharide (LOS) were phagocytosed by neutrophils in the absence of antibody and active complement. Confocal microscopy confirmed that the organisms were internalized by neutrophils. This susceptibility was not restricted to carrier isolates, since two of the strains were cultured from blood or cerebrospinal fluid. All four strains expressed Opa protein and had relatively less endogenous LOS and capsule sialylation compared to six strains that were resistant to this type of phagocytosis. Nonopsonic phagocytosis of two of the four strains was inhibited by exogenous sialylation of LOS LNnT and the binding of monoclonal antibody to LNnT. However, an isogenic mutant that lacked the LNnT structure was fully susceptible to nonopsonic phagocytosis. We conclude that group C meningococci can be phagocytosed by neutrophils in the absence of antibody and active complement possibly by two different mechanisms. Expression of Opa protein and downregulation of endogenous surface sialic acids analogous to what is seen for N. gonorrhoeae might be necessary for N. meningitidis as well.

Adult↗

PubMed1998

Neisseria gonorrhoeae induces focal polymerization of actin in primary human urethral epithelium.

The pathogenic Neisseria species induce cytoskeletal reorganization in immortalized cell lines. In Chang conjunctival epithelium and T84 intestinal epithelium, focal cytoskeletal rearrangements in which bacteria contacted the epithelial surface were observed. We show that actin footprints are induced in gonococcus-challenged primary urethral epithelium. Moreover, the microbes induced microvillus extension from the epithelial cell surface. Our results indicate that formation of actin footprints is not an artifact of commonly used immortalized cell lines.

Actins↗

PubMed1997

Evaluation of the virulence of nontypeable Haemophilus influenzae lipooligosaccharide htrB and rfaD mutants in the chinchilla model of otitis media.

Considerable evidence has implicated nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, two NTHi LOS-deficient mutants have been created and described. Strain 2019-DK1, an rfaD gene mutant, expresses a truncated LOS consisting of only three deoxy-D-manno-octulosonic acid residues, a single heptose, and lipid A. Strain 2019-B29, an isogenic htrB mutant, possesses an altered oligosaccharide core and an altered lipid A. Each strain's ability to colonize the nasopharynx and to induce OM subsequent to transbullar inoculation was evaluated in the chinchilla model. Nasopharyngeal colonization data indicate that the parent strain and both mutants are able to colonize the nasopharynx and exhibit comparable clearance kinetics. Compared with the parent and each other, however, the mutants demonstrated marked differences in virulence regarding their relative abilities to induce OM and persist in the middle ear post-transbullar inoculation. Strain B29 required a 3-log-greater dose to induce OM than the parent strain and did not exhibit evidence of sustained multiplication but persisted for the same duration as the parent. Conversely, strain-DK1, even when inoculated at a dose 4 logs greater than the parent dose, was eliminated from the middle ear 72 h after challenge. A comparison of the relative pathogenicities of these isolates provides the opportunity to address fundamental questions regarding the contribution of LOS to pathogenesis issues at the molecular level. Specifically, the impact of these LOS gene disruptions on OM pathogenesis can be defined and may thus provide potential new targets for future protection and intervention strategies.

Animals↗

PubMed1997

Phase variation and conservation of lipooligosaccharide epitopes in Haemophilus somnus.

The bovine-specific pathogen Haemophilus somnus is capable of undergoing structural and antigenic phase variation in its lipooligosaccharide (LOS) components after in vivo and in vitro passage. However, commensal isolates from the reproductive tract have not been observed to vary in phase (T. J. Inzana, R. P. Gogolewski, and L. B. Corbeil, Infect. Immun. 60:2943-2951, 1992). We now report that specific monoclonal antibodies (MAbs) to the LOSs of Haemophilus aegyptius, Neisseria gonorrhoeae, and Haemophilus influenzae, as well as H. somnus, reacted with some phase-variable epitopes in H. somnus LOS. All reactive MAbs bound to LOS components of about 4.3 kDa in the same H. somnus isolates, including a non-phase-varying strain. Following in vitro passage of a clonal variant of strain 738 that was nonreactive with the MAbs, 11.8% of young colonies shifted to a reactive phenotype. A digoxigenin-labelled 5'-CAATCAATCAATCAATCAATCAATCAAT-3' oligonucleotide probe hybridized to genomic DNA from strain 738 but did not react with DNA from a non-phase-varying strain. Sequence analysis of the gene containing 5'-CAAT-3' tandem sequences revealed 48% amino acid homology with the lex-2B gene-encoded protein of H. influenzae type b. Our results indicate that some LOS epitopes are conserved between H. somnus and other Haemophilus and Neisseria species, that LOS phase variation may occur at a high rate in some strains of H. somnus, and that phase variation may, in part, be due to 5'-CAAT-3' tandem sequences present in H. somnus genes.

Amino Acid Sequence↗

PubMed1997

Study of the role of the htrB gene in Salmonella typhimurium virulence.

We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.

Animals↗

PubMed1997

Identification of the ADP-L-glycero-D-manno-heptose-6-epimerase (rfaD) and heptosyltransferase II (rfaF) biosynthesis genes from nontypeable Haemophilus influenzae 2019.

Haemophilus influenzae is an important human pathogen. The lipooligosaccharide (LOS) of H. influenzae has been implicated as a virulence determinant. To better understand the assembly of LOS in nontypeable H. influenzae (NtHi), we have cloned and characterized the rfaD and rfaF genes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-epimerase and heptosyltransferase II enzymes, respectively. This cloning was accomplished by the complementation of Salmonella typhimurium lipopolysaccharide (LPS) biosynthesis gene mutants. These deep rough mutants are novobiocin susceptible until complemented with the appropriate gene. In this manner, we are able to use novobiocin resistance to select for specific NtHi LOS inner core biosynthesis genes. Such a screening system yielded a plasmid with a 4.8-kb insert. This plasmid was able to complement both rfaD and rfaF mutants of S. typhimurium. The LPS of these complemented strains appeared identical to the wild-type Salmonella LPS. The genes encoding the rfaD and rfaF genes from NtHi 2019 were sequenced and found to be similar to the analogous genes from S. typhimurium and Escherichia coli. The rfaD gene encodes a polypeptide of 35 kDa and the rfaF encodes a protein of 39 kDa, as demonstrated by in vitro transcription-translation studies. Isogenic mutants which demonstrated truncated LOS consistent with inner core biosynthesis mutants were constructed in the NtHi strain 2019. Primer extension analysis demonstrated the presence of a strong promoter upstream of rfaD but suggested only a very weak promoter upstream of rfaF. Complementation studies, however, suggest that the rfaF gene does have an independent promoter. Mass spectrometric analysis shows that the LOS molecules expressed by H. influenzae rfaD and rfaF mutant strains have identical molecular masses. Additional studies verified that in the rfaD mutant strain, D-glycero-D-manno-heptose is added to the LOS molecule in place of the usual L-glycero-D-manno-heptose. Finally, the genetic organizations of the inner core biosynthesis genes of S. typhimurium, E. coli, and several strains of H. influenzae were examined, and substantial differences were uncovered.

Base Sequence↗

PubMed1997

Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.

Asialoglycoprotein Receptor↗

PubMed1997

Characterization of a transposon Tn916-generated mutant of Haemophilus ducreyi 35000 defective in lipooligosaccharide biosynthesis.

To define the role of the surface lipooligosaccharide (LOS) of Haemophilus ducreyi in the pathogenesis of chancroid, Tn916 mutants of H. ducreyi 35000 defective in expression of the murine monoclonal antibody (MAb) 3F11 epitope on H. ducreyi LOS were identified by immunologic screening. One mutant, designated 1381, has an LOS which lacks the MAb 3F11 epitope and migrates with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene disrupted by the Tn916 element in strain 1381 was identified by cloning the sequences flanking the Tn916 element. The sequences were then used to probe a lambda DASHII genomic library. In strain 1381, Tn916 interrupts a gene which encodes an open reading frame (ORF) with an Mr of 40,246. This ORF has homology to the product of the rfaK gene of Escherichia coli. The major LOS glycoform produced by strain 1381 was analyzed by using a combination of mass spectrometry, linkage and composition analysis, and 1H nuclear magnetic resonance spectroscopy. The major LOS species was found to terminate in a single glucose attached to the heptose (L-glycero-D-manno-heptose, or Hep) trisaccharide core. In the wild-type strain 35000, glucose serves as the acceptor for the addition of the D-glycero-D-manno-heptose (or DDHep), which extends to form the mature branch of the H. ducreyi LOS. This mature oligosaccharide is in turn partially capped by the addition of sialic acid (NeuAc), i.e., NeuAc2 alpha-->3Gal beta1-->4GlcNAc beta1-->3Gal beta1-->4DDHep alpha1-->6Glc beta1 (W. Melaugh et al., Biochemistry 33:13070-13078, 1994). Since this LOS terminates prior to the addition of the branch DD-heptose, this gene is likely to encode the D-glycero-D-manno-heptosyltransferase. Strain 1381 exhibits a significant reduction in adherence to and invasion of primary human keratinocytes. This defect was complemented by the cloned heptosyltransferase gene, indicating that the terminal portion of the LOS oligosaccharide plays an important role in adherence to human keratinocytes.

Bacterial Adhesion↗

PubMed1997

Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization.

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.

Acyltransferases↗

PubMed1996

Plasmids with erythromycin resistance and catechol 2,3-dioxygenase- or beta-galactosidase-encoding gene cassettes for use in Neisseria spp.

Four new plasmids containing the ermC' (encoding a methyltransferase which confers resistance to erythromycin), xylE-ermC' (xylE, encoding catechol 2,3-dioxygenase) and lacZ-ermC' cassettes have been constructed. The 10-bp gonococcal uptake sequence has been placed downstream from ermC' to facilitate the delivery of these cassettes into pathogenic Neisseria spp. Several restriction sites have been placed to flank the cassettes to allow their excision and directional cloning. These plasmids will provide valuable tools for constructing insertional mutants and transcriptional fusions in Neisseria spp. or other bacteria.

Anti-Bacterial Agents↗

PubMed1996

Characterization of two transposon mutants from Haemophilus influenzae type b with altered lipooligosaccharide biosynthesis.

Two isogenic mutants of Haemophilus influenzae type b (Hib) strain A2 were prepared by random m-Tn3(Cm) insertions into the 7.4-kb lsg (lipooligosaccharide synthesis genes) region of Hib DNA, which consists of seven complete and one partial open reading frame (orfs). Compared to the parent A2 strain which produces a complex mixture of lipooligosaccharides (LOS), the mutant strains 281.25 and 276.4 produced only a few LOS species. The precise locations of transposon insertions into the lsg loci of these mutants were determined (base 3546 in orf 4 for strain 281.25 and base 4402 in orf 5 for strain 276.4), and the effects of these mutations on LOS biosynthesis and epitope expression were evaluated. When the O-deacylated LOS were analyzed by mass spectrometry, both strains contained major LOS species of M(r) 2601, 2439, and 2277, which consisted of a common heptose trisaccharide core structure [Hep3(PEA)Kdo(P)-lipid A, where Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-octulosonic acid, and PEA is phosphoethanolamine] and four, three, or two hexoses, respectively. These species represent the smallest components of the wild-type LOS mixture. The major LOS oligosaccharide obtained from the strain 281.25 by mild acid hydrolysis was dephosphorylated and shown by composition analysis, methylation analysis, mass spectrometry, and 2D NMR studies to be a triantennary structure consisting of a heptose trisaccharide core with two glucose disaccharide branches: Hep alpha 1 --> (Glc beta 1 --> 4Glc alpha 1 --> 3) 2Hep alpha 1 --> (Glc beta 1 --> 4Glc beta 1 --> 4)3Hep alpha 1 --> anhydroKdo. Unlike the parent A2 strain, mutant strain 281.25 cannot add galactoses to the branches of this octasaccharide. Strain 276.4 is similarly deficient, except that it can still utilize a minor biosynthetic pathway leading to the addition of sialyl-N-acetyllactosamine.

Antigens, Bacterial↗

PubMed1996

The pathogenesis of gonococcal urethritis in men: confocal and immunoelectron microscopic analysis of urethral exudates from men infected with Neisseria gonorrhoeae.

Confocal and immunoelectron microscopic analysis of urethral exudates from 12 men with gonococcal urethritis showed that Neisseria gonorrhoeae can invade urethral epithelial cells. Studies with acridine orange stain demonstrated that the majority of organisms within urethral epithelial cells were viable at the time of fixation. Three-dimensional modeling of an infected epithelial cell using image analysis of 21 digitized confocal sections stained with YOYO-1 and DiIC 18(3) revealed that gonococcal invasion of these cells occurred in a polar fashion, most likely at the epithelial luminal surface. Serial immunoelectron micrographs showed evidence of membrane fusion with pedestal formation between the gonococcus and the epithelial cell, gonococci within vacuoles, and occasional gonococci free in the cytoplasm. Immunoelectron microscopy studies showed ruptured vacuoles at the cell surface releasing organisms. These studies demonstrate that urethral epithelial cells are invaded by gonococci during the course of infection in males.

Epithelium↗