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.

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Haemophilus ducreyi is a sexually transmitted pathogen that colonizes the genital epithelium in humans, causing genital ulcers or chancroid. Its surface lipooligosaccharides (LOSs) have been shown to play a role in ulcer formation and may also be important in cell adhesion and invasion of host tissue. Earlier we presented a preliminary structure of the major LOS from strain 35000 that suggested the presence of terminal lactosamine [Melaugh, W., Phillips, N.J., Campagnari, A.A., Karalus, R., & Gibson, B. W. (1992) J. Biol. Chem. 267, 13434-13439]. We have now confirmed this structure and assigned the anomeric linkages by 2D NMR studies. In addition to this major structure, analysis by electrospray ionization mass spectrometry of both O-deacylated LOSs and the oligosaccharides released after treatment with mild acid indicates the presence of several other LOS glycoforms. These glycoforms constitute a series of both truncated and elongated analogs of the major oligosaccharide determined by NMR. One of these glycoforms exists as a smaller oligosaccharide corresponding to the major structure minus terminal galactose. Three other glycoforms appear as larger molecular weight species formed by the addition of phosphoethanolamine, N-acetylhexosamine, and N-acetylhexosamine plus hexose. Two sialylated glycoforms were also identified and subsequently confirmed by treatment with neuraminidase, but these glycoforms were not found in the released oligosaccharide pool due to the acid lability of of sialic acid. This study clearly indicates that the LOSs from H. ducreyi strain 35000 exist as a heterogeneous population whose structures differ primarily in their phosphorylation states and terminal sugars and whose terminal glycan structures can resemble those of human antigens.

The major lipooligosaccharides of the sexually transmitted pathogen Haemophilus ducreyi 35000 have been previously found to terminate in N-acetyllactosamine and sialyl-N-acetyllactosamine, Neu5Ac␣233Gal␤13 4GlcNAc (W. Melaugh, N. J. Phillips, A. A. Campagnari, M. V. Tullius, and B. W. Gibson, Biochemistry 33: 13070-13078, 1994). In this study, mass spectrometry and composition analyses have shown that the lipooligosaccharides from three other H. ducreyi strains also contain N-acetyllactosamine and are highly sialylated (Ϸ30 to 50%), although one African strain was found to contain neither of these structural features.The lipooligosaccharides (LOS) of Haemophilus ducreyi, like LOS of the mucosal pathogens Haemophilus influenzae and Neisseria gonorrheae, consist of a lipid A moiety embedded in the outer membrane of the bacterium attached to a variable oligosaccharide (13,14,21). It is thought that LOS is responsible for much of the cytotoxicity of H. ducreyi infection (4), but the role of the variable oligosaccharide has been difficult to define. Electrophoretic and mass spectrometric studies of LOS from numerous H. ducreyi strains have shown them to be highly heterogeneous (3,7,14). Previously, we had determined the structure of the major oligosaccharide from the LOS of H. ducreyi 35000 (14) and found it to contain terminal N-acetyllactosamine, i.e., Gal␤134GlcNAc, which is modified in some LOS by sialic acid. Given the importance of sialic acid in many biological systems (19), this acidic sugar is likely to play an important role in the pathology of H. ducreyi infection.In this study, we have investigated the presence or absence of N-acetyllactosamine and sialic acid in the LOS from H. ducreyi 188, NYC23, 233, and ATCC 33921 as well as one pyocin variant of strain 188 (2). Since N-acetyllactosamine is thought to be the acceptor for sialic acid (14), two intriguing questions were asked: do strains whose LOS terminate in Nacetyllactosamine also contain sialic acid, and do those strains whose LOS do not contain this disaccharide also lack sialic acid?Materials. Glucose (Glc), galactose (Gal), glucosamine (GlcN), galactosamine, 3-deoxy-D-manno-octulosonic acid (Kdo), neuraminidase (from Clostridium perfringens), and anhydrous hydrazine were all obtained from Sigma (St. Louis, Mo.). Aqueous HF (48%) was purchased from Mallinckrodt (Muskegon, Mich.), and sodium borodeuteride (98% deuterium) was obtained from Aldrich. Acetonitrile, water, and methanol were obtained from Burdick and Jackson (Muskegon, Mich.). Acetic anhydride was purchased from Supelco (Bellefonte, Pa.); methyliodide was purchased from Fluka (Buchs, Switzerland).Methods. The LOS from H. ducreyi 35000, 188, 233, NYC23, 188-2, and 33921 were isolated by using a modified phenolwater extraction procedure (22). LOS samples were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on a 14% polyacrylamide gel as previously described (14). Approximately 5 to 10 g of LOS was loaded into each well, and the LOS in even-numbered lanes ...

Previously, we reported the expression of chimeric lipopolysaccharides (LPS) in 5, 2475-2480). In this current study, we have analyzed the O-deacylated LPS and free oligosaccharides from three transformants (designated pGEMLOS-4, pGEM-LOS-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 Gal13 3GlcNAc13 is added, and in pGEMLOS-5, the structure is extended to Gal134GlcNAc133Gal133GlcNAc13. 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.Capsular strains of Haemophilus influenzae type b (Hib) 1 are responsible for various invasive and bacteremic infections in humans, including meningitis and pneumonia. The surface lipooligosaccharides (LOS) of Hib are known to be important factors in microbial virulence and pathogenesis (1-3). Structural studies of Hib LOS from wild-type (4, 5) and mutant strains (6 -8) have shown that the LOS contains a conserved heptose trisaccharide core that can be extended with additional sugars on each heptose. However, efforts to correlate defined LOS structures with specific biological functions have been hindered by the high degree of microheterogeneity and antigenic variability of Hib LOS. Additionally, some Hib LOS epitopes phase vary at high frequencies due to slip-strand mispairing (9), adding a further level of structural complexity to wild-type Hib LOS populations.To facilitate the elucidation of LOS biosynthesis and to determine the relationship of LOS structures to the biology and pathophysiology of Haemophilus infections, our laboratory has cloned a gene cluster from Hib strain A2 containing LOS synthesis genes (lsg) (10). The lsg loci are contained within a 7.4-kb DNA fragment, consisting of seven complete open reading frames (ORFs) (11). This region is one of several distinct loci found in the genome sequence of Hib strain Rd (12) that have been associated with lipopolysaccharide (LPS) biosynthesis (13). Recently, a series of isogenic mutants of Hib A2 was generated by transposon mutagenesis of the lsg region using minitransposon m-Tn3(Cm) (14). Several of the transposon mutants produced much simpler LOS mixtures than the wildtype Hib A2 LOS and no longer reacted with one or more of the...

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-mannoheptose 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.

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Haemophilus ducreyi, a cause of genital ulcer disease in developing countries, appears to facilitate the heterosexual transmission of the human immunodeficiency virus in Africa. Despite an increase in studies of this gram-negative human pathogen, little is known about the pathogenesis of chancroid. Our studies have shown that the lipooligosaccharides (LOS) of H. ducreyi may play an important role in ulcer formation. Monoclonal antibody and mass spectrometric analyses identified a terminal trisaccharide present on H. ducreyi LOS that is immunochemically similar to human paragloboside. This epitope is present on the LOS of Neisseria gonorrhoeae, and it may be the site of attachment for pyocin lysis. We have used pyocin, produced by Pseudomonas aeruginosa, to select LOS variants with sequential saccharide deletions from N. gonorrhoeae. On the basis of the similarities between N. gonorrhoeae and H. ducreyi LOS, we employed the same technique to determine if H. ducreyi strains were susceptible to pyocin lysis. In this study, we report the generation of a pyocin N-resistant H. ducreyi strain which synthesizes a truncated version of the parental LOS. Further studies have shown that this H. ducreyi variant has lost the terminal LOS epitope defined by monoclonal antibody 3F11. This report demonstrates that H. ducreyi is sensitive to pyocins and that this technique can be used to generate H. ducreyi LOS variants. Such variants could be used in comparative studies to relate LOS structure to biologic function in the pathogenesis of chancroid.