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M A Apicella

Publications and source records attributed to M A Apicella.

At least 73 records · Page 4Linked to original sources
PubMed1993

Structural studies of the lipooligosaccharides from Haemophilus influenzae type b strain A2.

The outer membrane lipooligosaccharides (LOS) from Haemophilus influenzae type b strain A2 are a heterogeneous mixture of glycolipids containing a conserved Lipid A structure and a variable oligosaccharide moiety. After O-deacylation by treatment with anhydrous hydrazine, the O-deacylated LOS mixture was analyzed by electrospray mass spectrometry and shown to contain 11 components, ranging in M(r) from 2277.8 to 3416.4. The majority of these structures contained a variable number of hexoses, three L-glycero-D-manno-heptoses, and one 3-deoxy-D-manno-octulosonic acid (KDO) residue attached to a diphosphorylated O-deacylated Lipid A moiety. Additional phosphate and phosphoethanolamine (PEA) groups were also present on the oligosaccharide structures. Two minor high molecular weight components were also observed that contained N-acetylhexosamine and sialic acid. Neuraminidase treatment of the O-deacylated LOS mixture resulted in the loss of sialic acid from these latter two species. After mild acid hydrolysis and separation by size-exclusion chromatography, liquid secondary ion mass spectrometry identified six major and four minor oligosaccharides, ranging in M(r) from 1243.4 to 2215.8. These released oligosaccharides contained a common heptose trisaccharide core structure with anhydro-KDO at the reducing terminus, which arises as an artifact of the hydrolysis procedure by beta-elimination of a phosphate group from the 4-position of KDO. Selected oligosaccharide fractions were subjected to composition and methylation analyses and sequenced by tandem mass spectrometry. Taken together, these data defined the major O-deacylated LOS as follows: [formula: see text] Higher molecular weight structures in the mixture contained galactose, N-acetylglucosamine, and sialic acid as additional branch sugars, suggesting that H. influenzae A2 is capable of forming a sialylated lactosamine structure.

Carbohydrate Sequence↗
PubMed1993

Lipo-oligosaccharides (LOS) of mucosal pathogens: molecular mimicry and host-modification of LOS.

Immunochemical studies of the lipo-oligosaccharides (LOS) of the Gram-negative bacteria Neisseria gonorrhoeae and Neisseria meningitidis have revealed some interesting structural characteristics of these LOS that might relate to their roles during pathogenesis. The carbohydrate moieties of the LOS of pathogenic Neisseria mimic carbohydrates present in glycosphingolipids of human cells. Firstly, an LOS component present among a number of Neisseria species is antigenically and/or chemically identical to lactoneoseries glycosphingolipids present in human cells. The lactoneoseries LOS becomes sialylated on Neisseria gonorrhoeae when they are grown in the presence of cytidine 5'-monophospho-N-acetyl-neuraminic acid (CMP-NANA), the nucleotide sugar for sialic acid. Examination of gonococci present in exudates from males with natural infection indicates that sialylation also occurs in vivo. The mechanism for this process apparently involves a bacterial sialyltransferase scavenging available host CMP-NANA ("host-modification" of LOS) and transferring the sialic acid to the lactoneoserieslike LOS. Strains of N. meningitidis and Haemophilus influenzae also express similarly sialylated LOS suggesting that this is a common mechanism of pathogenesis among these bacteria. Additional examples of LOS that mimic other glycosphingolipid series have been identified also and the fact that multiple series can be expressed in a single population of gonococci suggests that a diverse set of LOS can be presented to the host during infection. It is possible that this diverse set of LOS serve different functions for the bacteria in various hosts and/or environments during infection.

Antigens, Bacterial↗
PubMed1993

Cloning of a gonococcal DNA sequence that complements the lipooligosaccharide defects of Neisseria gonorrhoeae 1291d and 1291e.

An isogenic set of gonococcal lipooligosaccharide (LOS) mutants derived from pyocin treatment of Neisseria gonorrhoeae 1291 was used to identify cloned gonococcal DNA fragments. A gene bank from N. gonorrhoeae 1291c chromosomal DNA was made in pLEE10, a shuttle vector that replicates in the gonococcus and Escherichia coli. A plasmid (pSG30) that could transform the LOS mutants 1291d and 1291e to reactivity with monoclonal antibody 3F11 and to production of an LOS component with migration identical to that of the parent, 1291, was identified. pSG30 contains a 9-kb EcoRI fragment. Curing studies indicate that pSG30 encodes gene products that affect LOS biosynthesis in trans. Subcloning identified a 2.6-kb HincII fragment (pSG38) that retained the ability to modify the LOS of 1291d and 1291e. The DNA regions involved in modification of 1291d and 1291e were named lsi-4 and lsi-5, respectively. The region of pSG38 that was involved in LOS modification was further localized by the construction of exonuclease III deletion plasmids. Transformation of these constructs identified a 750-bp fragment that retains the ability to modify 1291e and a 540-bp fragment which retains the ability to modify 1291d.

Base Sequence↗
PubMed1993

Investigation of the structural heterogeneity of lipooligosaccharides from pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry.

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophilus and Neisseria species is evident from the multiplicity of components observed with electrophoretic analyses. Knowledge of the precise structures that make up these diverse LOS molecules is clearly the key to reaching an understanding of pathogenic processes such as phase variation and molecular mimicry. Except for a few cases, little is known about the specific structural features of LOS that underlie phase variation and molecular mimicry, partly because of the inherent difficulties in the structural elucidation of these complex glycolipids. In the lipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia coli, rough, or R-type, mutants have been isolated that have provided insight into the biosynthetic pathways and associated genetics that control LPS expression. Nonetheless, recent work has shown that these R-type LPS are more complex than originally thought, and significant heterogeneity is still observed, primarily in their phosphorylation states. In order to investigate the structures of LPS and LOS in a more rapid fashion, we have determined the precise molecular weights of LOS (and LPS) preparations from various Haemophilus, Neisseria, and Salmonella species by electrospray ionization-mass spectrometry. The LOS (or LPS) were first O-deacylated under mild hydrazine conditions to remove O-linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M-nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Mass spectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heterogeneity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the variable phosphorylation of the core of S. typhimurium LPS have afforded insights into the biosynthetic pathways used by these organisms. Information of this type is important for understanding the underlying genetic and environmental factors controlling LOS and LPS expression.

Antigens, Bacterial↗
PubMed1992

Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae.

Oligosaccharides released from the lipooligosaccharides (LOS) of Haemophilus influenzae nontypable strain 2019 by mild acid hydrolysis were fractionated by size exclusion chromatography and analyzed by liquid secondary ion mass spectrometry. The major component of the heterogeneous mixture was found to be a hexasaccharide of Mr 1366, which lost two phosphoethanolamine groups upon treatment with 48% aqueous HF. The dephosphorylated hexasaccharide was purified and shown by tandem mass spectrometry, composition analysis, methylation analysis, and two-dimensional nuclear magnetic resonance studies to be Gal beta 1----4Glc beta 1----(Hep alpha 1----2Hep alpha 1----3) 4Hep alpha 1----5anhydro-KDO, where Hep is L-glycero-D-manno-heptose and KDO is 3-deoxy-D-manno-octulosonic acid. An analogous structure containing authentic KDO was generated from LOS that had been HF-treated prior to acetic acid hydrolysis, suggesting that the reducing terminal anhydro-KDO moiety is produced as an artifact of the hydrolysis procedure by beta-elimination of a phosphate substituent from C-4 of KDO. Mass spectral analyses of O-deacylated LOS and free lipid A confirmed that, in addition to the two phosphoethanolamines on the oligosaccharide and two phosphates on the lipid A, another phosphate group exists on the KDO. This KDO does not appear to be further substituted with additional KDO residues in intact H. influenzae 2019 LOS. The terminal disaccharide epitope, Gal beta 1----4Glc beta 1----, of the hexasaccharide is also present on lactosylceramide, a precursor to human blood group antigens. It is postulated that the presence of this structure on H. influenzae LOS may represent a form of host mimicry by the pathogen.

Antigens, Bacterial↗
PubMed1992

A nontoxic, idiotope vaccine against gram-negative bacterial infections.

Experiments were performed to test the ability of mouse antiidiotopic mAb, specific for an antilipid A mAb, to act as a vaccine against gram-negative bacterial infections. Lipid A is a conserved region of bacterial LPS. Immunization with the antiidiotopic antibodies, coupled to an immunogenic carrier protein (hemocyanin), specifically induced anti-LPS antibody responses in animals from different species. In a mouse model, this immunization resulted in protection against both lethal gram-negative bacteremia and endotoxemia. The antiidiotopic antibodies, however, did not stimulate endotoxin-associated bioactivities, such as induction of TNF and IL-1. These results support the hypothesis that an idiotope vaccine can stimulate beneficial protective immunity against gram-negative infections without the toxicity inherent in LPS.

Animals↗
PubMed1992

Outer membrane protein P6 of Haemophilus influenzae binds to its own gene.

Outer membrane protein P6 is an important antigen expressed on the surface of all strains of Haemophilus influenzae. The predicted amino acid sequence of P6 contains a region of alpha helices that shares sequence identity with a family of helix-turn-helix DNA-binding proteins. A search for sequence-specific binding sites that resemble an operator region within the gene revealed a sequence with striking homology to the consensus operator sequence for lambda Cro and repressor. To test the hypothesis that P6 binds its own gene, purified P6 on nitrocellulose was probed with plasmid DNA containing the P6 gene. P6 bound the P6 gene in this Southwestern blot assay. To further test the observation, gel shift analysis was performed. Gel shift assays using a P6-specific monoclonal antibody demonstrated that P6 in crude cell extracts binds to the region of the gene containing the putative binding site. Competition with a synthetic oligonucleotide corresponding to the putative binding site inhibited binding of P6 to the P6 gene on nitrocellulose and in the gel shift assay. In addition, this oligonucleotide bound directly to P6 on nitrocellulose. Finally, DNase footprinting confirmed that P6 bound specifically to the same region of the P6 gene. These results indicate that P6 binds to a sequence-specific site within its own gene, suggesting that P6 regulates its own expression. This represents the first example of a Gram-negative outer membrane protein binding to its own gene and has potentially important implications as a mechanism for regulation of expression of outer membrane antigens.

Amino Acid Sequence↗
PubMed1992

Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated.

The lipooligosaccharides (LOS) of strains of Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica contain epitopes that are antigenically and structurally similar to carbohydrates present in human glycosphingolipids. LOS from strains of Haemophilus influenzae and H. influenzae biogroup aegyptius were tested for the binding of monoclonal antibodies (MAbs) that bind to human glycosphingolipids possessing Gal beta 1-4GlcNAc (MAb 3F11) and Gal alpha 1-4Gal beta 1-4Glc (MAb anti-Pk). In solid-phase radioimmunoassays, the LOS of 18 of 19 H. influenzae type b (Hib), 8 of 19 nontypeable H. influenzae, and 10 of 20 H. influenzae biogroup aegyptius strains bound MAb anti-Pk. The LOS of 13 of 19 Hib, 10 of 16 nontypeable H. influenzae, and 2 of 18 H. influenzae biogroup aegyptius strains bound MAb 3F11. Neuraminidase treatment of the strains increased the binding of MAb 3F11 by more than twofold in 47% of the H. influenzae strains, suggesting that sialic acid occluded the LOS structure recognized by MAb 3F11. The material released from neuraminidase-treated Hib LOS was confirmed to be sialic acid by high-performance anion-exchange chromatography. A recombinant plasmid containing genes involved in Hib LOS biosynthesis directed the expression (assembly) of the 3F11 epitope in Escherichia coli. These studies demonstrate that H. influenzae and H. influenzae biogroup aegyptius express at least two LOS epitopes that are similar to those present in human glycosphingolipids. Sialic acid was present on the LOS of some H. influenzae strains and prevented the binding of MAb 3F11 to its epitope. The oligosaccharide portion of sialylated LOS may also resemble sialylated oligosaccharides present in human glycosphingolipids (gangliosides).

Antibodies, Monoclonal↗
PubMed1992

Generation of lipooligosaccharide mutants of Haemophilus influenzae type b.

We previously reported the analysis of recombinant plasmids from Haemophilus influenzae type b (Hib) that lead to modifications of Escherichia coli lipopolysaccharide (LPS) (Y. Abu Kwaik, R. E. McLaughlin, M. A. Apicella, and S. M. Spinola, Mol. Microbiol. 5:2475-2480, 1991). The modified LPS species are recognized by monoclonal antibodies (MAbs) 6E4 and 3F11. MAb 6E4 binds to a stable 2-keto-3-deoxyoctulosonic acid epitope, while MAb 3F11 binds to a Gal beta 1-4GlcNac epitope that phase varies in Hib at a frequency of 2 to 5%. The internal EcoRI fragment containing most of the DNA required for LPS modification in E. coli was used as the target for transposon mutagenesis. Plasmids containing minitransposon m-Tn3(Cm) randomly inserted into the target fragment were transformed into the isogenic Hib strain, and transposon integration into the Hib chromosome was verified by colony hybridization. The lipooligosaccharides of 36 transformants were phenotypically and antigenically characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity with a variety of MAbs that recognize both stable and phase-varying lipooligosaccharide epitopes. The majority of the mutants had altered reactivity with MAb 6E4. With one exception, these mutants retained the ability to express phase-varying epitopes. Analysis of the transformants suggested that the 6E4 epitope was contained on an oligosaccharide chain separate from that of phase-varying epitopes and appeared to be assembled in at least three separate steps.

Antigens, Bacterial↗
PubMed1991

Expression of paragloboside-like lipooligosaccharides may be a necessary component of gonococcal pathogenesis in men.

To learn how lipooligosaccharide (LOS) phase variations affect pathogenesis, we studied two male volunteers who were challenged intraurethrally with Neisseria gonorrhoeae that make a single LOS of 3,600 daltons and sequentially followed LOS expression by gonococci as urethritis developed. LOS variation occurred in vivo. Signs and symptoms of gonorrhea began with the appearance of variants making 4,700-dalton LOS that are immunochemically similar to glycosphingolipids of human hematopoietic cells (Mandrell, R.E., J.M. Griffiss, and B.A. Macher. 1989. J. Exp. Med. 168:107) and that have acceptors for sialic acid. A variant that appeared at the onset of leukorrhoea was shed by 34/36 men with naturally acquired gonorrhea at the time they sought medical attention; the other two shed the variant associated with dysuria. None shed the challenge variant. These data show that in vivo phase shifts to higher molecular mass LOS that mimic human cell membrane glycolipids are associated with the development of gonococcal leukorrhea.

Carbohydrate Sequence↗
PubMed1991

The structural basis for pyocin resistance in Neisseria gonorrhoeae lipooligosaccharides.

Pyocin resistance in a strain of Neisseria gonorrhoeae has been found to be associated with structural differences in the oligosaccharide moieties of the gonococcal outer membrane lipooligosaccharides (LOS). N. gonorrhoeae strain 1291 had been treated with several pyocins, usually lethal bacteriocins produced by Pseudomonas aeruginosa, and a series of surviving mutants were selected. The LOS of these pyocin-resistant mutants had altered electrophoretic mobilities in sodium dodecyl sulfate-polyacrylamide gels (Dudas, K. C., and Apicella, M. A. (1988) Infect. Immun. 56, 499-504). Structural analyses of the oligosaccharide portions of the wild-type (1291 wt) and five pyocin-resistant strains (1291a-e) by liquid secondary ion mass spectrometry, tandem mass spectrometry, and methylation analysis revealed that four of the mutant strains make oligosaccharides that differ from the wild-type LOS by successive saccharide deletions (1291a,c-e) and, in the oligosaccharide of 1291b, by the addition of a terminal Gal to the 1291c structure. The composition, sequence, and linkages of the terminal tetrasaccharide of the wild-type LOS are the same as the lacto-N-neotetraose terminus of the human paragloboside (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-ceramide), and both glycolipids bound the same monoclonal antibodies O6B4/3F11 that recognize this terminal epitope. None of the pyocin-resistant mutants bound this antibody. The 1291b LOS bound a monoclonal antibody that is specific for Gal alpha 1----4Gal beta 1----4Glc-ceramide (Pk glycosphingolipid) and shared a common composition, sequence, and linkages with this latter glycosphingolipid. Organisms that bound the anti-Pk monoclone occurred at the rate of approximately 1/750 among the wild-type parent strain. This structural information supports the conclusion that treatment with pyocin selects for mutants with truncated LOS structures and suggests that the oligosaccharides contained in the LOS of the wild-type strain and 1291b mimic those of human glycosphingolipids.

Carbohydrate Sequence↗
PubMed1991

The primary B cell response to the O/core region of bacterial lipopolysaccharide is restricted to the Ly-1 lineage.

Experiments were performed to test the hypothesis that Ly-1 B cells respond to antigenic challenge with LPS from gram-negative bacteria. To perform these experiments, the splenic fragment culture system for the study of B cell precursors was used. We found that a significant number of anti-O/core, but not anti-lipid A, precursors expressed the lambda L chain. A restriction of the anti-LPS response to Ly-1 B cells was tested using a Ly-1 depletion protocol. We found that the anti-O/core antibody response was restricted to the Ly-1 B cell lineage. In contrast, conventional B cells, not Ly-1 B cells, respond to an antigenic challenge with lipid A. Our results further support the idea that the Ly-1 B cell lineage serves a direct role in protecting against certain bacterial infections.

Animals↗
PubMed1991

Epitope expression and partial structural characterization of F62 lipooligosaccharide (LOS) of Neisseria gonorrhoeae: IgM monoclonal antibodies (3F11 and 1-1-M) recognize non-reducing termini of the LOS components.

F62 LOS of Neisseria gonorrhoeae consists of two components. The higher molecular weight (MW) component is recognized by monoclonal antibody (MAb) 1-1-M and the smaller MW component by MAb 3F11. Epitope expression of the two LOS components and their partial structures were investigated by treating the F62 LOS with several glycosidases and then monitoring their antigenicity with the two mouse IgM MAbs. The 1-1-M-defined LOS component was cleaved with both beta-N-acetylhexosaminidase and endo-beta-galactosidase, and each cleavage resulted in the loss of expression of the 1-1-M-defined epitope. The N-acetylhexosamine (HexNAc) released by the hexosaminidase was found to be GalNAc, and the smaller oligosaccharide released by the endo enzyme was identified to be a dimer GalNAc beta----Gal. In contrast, the MAb 3F11-defined LOS component was not digested by the endo galactosidase, but it was cleaved with alpha and beta-galactosidase, and expression of the MAb 3F11-defined LOS epitope expression of the MAb 3F11-defined LOS was abolished by the treatment with each of two exo enzymes. MAb 3F11 bound to the 1-1-M-defined LOS component resulting from the removal of the beta-GalNAc residue, and the resulting LOS was further cleaved with beta-galactosidase, but not with alpha-galactosidase. From these results, we conclude the following: (1) MAbs 1-1-M and 3F11 both recognize the non-reducing termini of the LOS components; (2) the 1-1-M-defined LOS component has the GalNAc beta----Gal beta 1----4-Glc (or GlcNAc) structure, and the GalNAc beta----Gal residue is involved in the MAb 1-1-M-defined epitope; (3) the MAb 3F11-defined LOS component may not have a Gal beta 1----4GlcNAc beta 1----4Gal beta 1----4Glc structure within the molecule. However, it has beta-Gal residue at its non-reducing terminus, and this residue is involved in the MAb 3F11-defined epitope; (4) the two LOS components share a similar antigenic structure, and the 3F11-defined epitope structure is present in the MAb 1-1-M-defined LOS component. Expression of this epitope within the 1-1-M-defined LOS molecule is blocked by the beta-GalNAc residue; however, the beta-GalNAc residue at the non-reducing end may be not the only structural difference between the two components.

Acetylglucosaminidase↗
PubMed1991

Analysis of Haemophilus influenzae type b lipooligosaccharide-synthesis genes that assemble or expose a 2-keto-3-deoxyoctulosonic acid epitope.

We recently isolated a recombinant phage from a Haemophilus influenzae type b (Hib) library that assembles an oligosaccharide with an apparent molecular weight of 1400 (1.4 K) on a 4.1 K Escherichia coli lipopolysaccharide (LPS) structure, producing a 5.5 K LPS species that contains a KDO (2-keto-deoxyoctulosonic acid) epitope. Subcloning and deletional analysis of the 14 kb Haemophilus insert showed that three overlapping restriction fragments contained within a 7.2 kb Pstl-BamHl fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5 K, 5.1 K and 5.5 K. Only the 5.5 K species contained the KDO epitope. We confirmed the relationship between the cloned genes and Hib lipooligosaccharide (LOS) biosynthesis by constructing a mutant that expressed an altered LOS. Thus, the Hib 7.2 kb Pstl-BamHl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.

Blotting, Western↗