Michael A. Apicella

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

ABSTRACT 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 α-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 α2,6-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 α2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

Haemophilus influenzae Type b Strain A2 Has Multiple Sialyltransferases Involved in Lipooligosaccharide Sialylation

The lipooligosaccharide (LOS) ofHaemophilus influenzae contains sialylated glycoforms, and a sialyltransferase, Lic3A, has been previously identified. We report evidence for two additional sialyltransferases, SiaA, and LsgB, that affect N-acetyllactosamine containing glycoforms. Mutations in genes we have designated siaA and lsgBaffected only the sialylated glycoforms containingN-acetylhexosamine. A mutation in siaA resulted in the loss of glycoforms terminating in sialyl-N-acetylhexosamine and the appearance of higher molecular weight glycoforms, containing the addition of phosphoethanolamine, N-acetylgalactosamine, andN-acetylneuraminic acid. Chromosomal complementation of thesiaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lackingN-acetylhexosamine and had no effect on sialylation of the terminal N-acetyllactosamine epitope. A double mutant insiaA and lic3A resulted in the complete loss of sialylation of the terminal N-acetyllactosamine epitope and expression of the higher molecular weight sialylated glycoforms seen in thesiaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction inN-acetyllactosamine containing glycoforms. A triple mutant of siaA, lic3A, and lsgB contained no sialylated glycoforms. These results demonstrate that the sialylation of the LOS of H. influenzae is a complex process involving multiple sialyltransferases.

Identification of a 16,600-dalton outer membrane protein on nontypeable Haemophilus influenzae as a target for human serum bactericidal antibody.

A 16,600-D outer membrane protein is present in all strains of Haemophilus influenzae and antibodies to this protein are present in human serum. This study was designed to assess the role of this outer membrane protein (P6) in nontypeable H. influenzae as a target for human serum bactericidal antibody. P6 was isolated and coupled to an affinity column. Depleting normal human serum of antibodies to P6 by affinity chromatography resulted in reduced bactericidal activity of that serum for nontypeable H. influenzae. Immunopurified antibodies to P6 from human serum were bactericidal. Finally, preincubation of bacteria with a monoclonal antibody that recognizes a surface epitope on P6, inhibited human serum bactericidal killing. Taken together, these experiments establish that P6 is a target for human bactericidal antibodies. This observation provides evidence that P6 plays a potentially important role in human immunity to infection by nontypeable H. influenzae.

Lipooligosaccharide epitopes shared among Gram-negative non-enteric mucosal pathogens

The non-enteric Gram-negative human pathogens, B. catarrhalis, H. ducreyi, H. influenzae, N. gonorrhoeae and N. meningitidis, do not have repeating O-antigens as part of their principle surface glycolipid, the lipooligosaccharide (LOS). Because they have similar LOS structures, we studied the conservation of LOS oligosaccharide epitopes among these organisms. Twenty-one monoclonal antibodies (mAbs) generated by immunizing mice with H. influenzae, N. gonorrhoeae and N. meningitidis were studied for cross reactivity. Five mAbs generated against non-typable H. influenzae were the only strain-specific antibodies. Ten mAbs reacted to LOS epitope(s) common to a genera or species, and six mAbs bound to epitope(s) on the LOS of strains from different genera. Some cross reactive mAbs bound to LOS bands of similar molecular weights, while others bound to bands of varying molecular weights. mAb 3F11, whose epitope mimics a human blood-group antigen, bound to a 4.8 kDa LOS band in N. gonorrhoeae and H. ducreyi, two pathogens that infect genital epithelium. mAb 3D9, whose epitope consists of 2-keto-3-deoxyoctulosonic acid (KDO), reacted with different LOS bands in N. gonorrhoeae, H. influenzae and some R mutants of S. minnesota. A 14 kb restriction fragment containing lipooligosaccharide synthesis genes responsible for the assembly of the 3D9 epitope in H. influenzae hybridized to all H. influenzae strains tested but did not hybridize to gonococcal and S. minnesota strains that expressed this epitope. These studies demonstrate that conserved LOS epitope(s) exist among different species and genera of non-enteric human pathogens and that different genetic mechanisms may have evolved in these pathogens to assemble some of these conserved epitopes.

Biofilm Formation by Neisseria gonorrhoeae

ABSTRACT 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 μM 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.

Lipooligosaccharide Pk (Galα1-4Galβ1-4Glc) Epitope of Moraxella catarrhalis Is a Factor in Resistance to Bactericidal Activity Mediated by Normal Human Serum

ABSTRACT 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 inM. 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 2951galE had lost two hexose residues due to thegalE mutation and that the resultant LOS structure lacked the (Galα1-4Galβ1-4Glc) Pk epitope found onM. catarrhalis 2951. Wild-type M. catarrhalis2951 is resistant to complement-mediated serum bactericidal activity. In contrast, a greater than 2-log10-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 log10 unit. These studies suggest that the Pkepitope of the LOS may be an important factor in the resistance of M. catarrhalis to the complement-mediated bactericidal effect of normal human serum.

Septicemia due to DF-2: Cause of a false-positive cryptococcal latex agglutination result

A previously healthy 26-year-old man presented with fever, headache, skin rash, and thrombocytopenia. Cultures of blood and cerebrospinal fluid yielded a fastidious gram-negative bacillus, identified as DF-2. A unique feature of this case was the presence of a false-positive latex agglutination result for cryptococcal antigen in the cerebrospinal fluid in the absence of pleocytosis. Additional laboratory studies, which included indirect immunofluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, however, failed to reveal common antigenic surface components between these organisms.

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.

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–Bam HI fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5K, 5.1K and 5.5K. Only the 5.5K 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‐Bam Hl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.

The thymus-dependent nature of the murine antibody response to a monoclonal anti-idiotypic antibody to the Neisseria meningitidis serogroup C capsular polysaccharide

Idiotype vaccines are proteins which may offer an alternative strategy for the conversion of a thymus-independent antigen into a thymus-dependent immunogen. To examine this question, we have studied the nature of the immune response to a monoclonal anti-idiotypic antibody, designated 6F9, which acts as a surrogate of Neisseria meningitidis serogroup C capsular polysaccharide, and compared this response to the nominal antigen, the meningococcal C-polysaccharide (MCP). BALB/c mice immunized with an optimal dose (100 micrograms) of 6F9 generate a specific anti-MCP IgG response which is maximal after 4 weeks. Secondary immunization with 6F9 results in a three- to five-fold increase in the specific IgG response. Mice given an optimal immunizing dose of MCP (5 micrograms) failed to generate an anti-MCP IgG response. No secondary response is detectable in mice immunized with MCP. Animals immunized with 6F9 and subsequently challenged with live meningococci group C show a significant anti-MCP IgG response. BALB/c nu/nu mice fail to generate an anti-MCP IgG antibody response to 6F9, while the nu/+ controls generate an anti-MCP IgG antibody titer 100 times that of the MCP-immunized mice. Neonatal mice that failed to respond to MCP developed early IgM and a subsequent IgG anti-MCP response after immunization with 6F9. These data demonstrate that the anti-idiotype 6F9, the combining site of which contains a surrogate image of the meningococcal group C capsular polysaccharide, evokes the responses expected of a T-dependent antigen.