Towards understanding the functional role of the glycosyltransferases involved in the biosynthesis of Moraxella catarrhalis lipooligosaccharide

The glycosyltransferase enzymes (Lgts) responsible for the biosynthesis of the lipooligosaccharide-derived oligosaccharide structures from Moraxella catarrhalis have been investigated. This upper respiratory tract pathogen is responsible for a spectrum of illnesses, including otitis media (middle ear infection) in children, and contributes to exacerbations of chronic obstructive pulmonary disease in elderly patients. To investigate the function of the glycosyltransferase enzymes involved in the biosynthesis of lipooligosaccharide of M. catarrhalis and to gain some insight into the mechanism of serotype specificity for this microorganism, mutant strains of M. catarrhalis were produced. Examination by NMR and MS of the oligosaccharide structures produced by double-mutant strains (2951lgt1/4Delta and 2951lgt5/4Delta) and a single-mutant strain (2951lgt2Delta) of the bacterium has allowed us to propose a model for the serotype-specific expression of lipooligosaccharide in M. catarrhalis. According to this model, the presence/absence of Lgt4 and the Lgt2 allele determines the lipooligosaccharide structure produced by a strain. Furthermore, it is concluded that Lgt4 functions as an N-acetylglucosylamine transferase responsible for the addition of an alpha-D-GlcNAc (1-->2) glycosidic linkage to the (1-->4) branch, and also that there is competition between the glycosyltransferases Lgt1 and Lgt4. That is, in the presence of an active Lgt4, GlcNAc is preferentially added to the (1-->4) chain of the growing oligosaccharide, instead of Glc. In serotype B strains, which lack Lgt4, Lgt1 adds a Glc at this position. This implies that active Lgt4 has a much higher affinity/specificity for the beta-(1-->4)-linked Glc on the (1-->4) branch than does Lgt1.     

The effects of S-carboxymethylcysteine and N-acetylcysteine on the adherence of Moraxella catarrhalis to human pharyngeal epithelial cells

We investigated the effects of two mucoregulating drugs, S-carboxymethylcysteine (S-CMC) and N-acetylcysteine (NAC), on the attachment of Moraxella catarrhalis (M. catarrhalis) to pharyngeal epithelial cells. The attachment of M. catarrhalis decreased (33-57%) significantly (P<0.01) in a dose-dependent manner in cells treated with mucoregulating drugs as compared to the control. There was a significant (P<0.01) decrease (35-45%) in the attachment of M. catarrhalis to pharyngeal cells after oral administration of S-CMC. By electron microscopic observation, it was found that there was a fine, granular, electron-dense, ruthenium red-positive layer on the surface of pharyngeal epithelial cells; this layer was absent on cell surfaces treated with mucoregulating drugs. Possibly, this layer contained the portion of M. catarrhalis receptor which is responsible for the attachment of this bacteria to pharyngeal epithelial cells. From the above results, it may be concluded that one of the mechanisms of mucoregulating drugs to decrease the episode of respiratory infections in patients with chronic respiratory diseases is by inhibiting the attachment of bacteria to the upper respiratory tract.     

Attachment of Moraxella catarrhalis to pharyngeal epithelial cells is mediated by a glycosphingolipid receptor

Abstract Moraxella catarrhalis is one of the major pathogens of respiratory infections and has the ability to attach to the pharyngeal cells via fimbriae. We characterized the epithelial cell receptor to which fimbriate M. catarrhalis binds. Neuraminidase pretreatment of pharyngeal epithelial cells resulted in a significant decrease of M. catarrhalis attachment, suggesting interaction with the sialic acid component. The attachment was not decreased in M. catarrhalis pretreated with 2 and 1 mg/ml of fucose, N -acetyl-neuraminic acid, N -acetyl-glucosamine, N -acetyl-galactosamine, acetyl-salicylic acid and colominic acid. However, M. catarrhalis treated with gangliosides M1, M2, D1a, D1b and T1a at a concentration of 2.5 μg/ml had significantly decreased the attachment compared to the control. In contrast treatment with gangliosides M3 and asialoganglioside M1 did not decrease the attachment of M. catarrhalis and thereby provided evidence for specificity of the inhibition. Concentration dependent effects of ganglioside M2 on the attachment were also observed. Other fimbriate isolates of M. catarrhalis showed decrease in attachment after treatment with ganglioside M2. However there was no effect on attachment when a nonfimbriate isolate was treated with ganglioside M2. This study indicates that the receptor of fimbriate M. catarrhalis on pharyngeal epithelial cells resides in the sequences of ganglioside M2.     

Sulfatide and its synthetic analogues recognition by Moraxella catarrhalis

Moraxella catarrhalis is one of the major pathogens of respiratory and middle ear infections. Attachment of this bacterium to the surface of human pharyngeal epithelial cells is the first step in the pathogenesis of infections. This study revealed that sulfatide might act as a binding molecule for the attachment of M. catarrhalis to human pharyngeal epithelial cells. Furthermore, six different synthetic sulfatides were found to inhibit the attachment of M. catarrhalis significantly at an optimum concentration of 10 microg/ml. Synthetic sulfatides may have the potential to be used as a therapy to prevent M. catarrhalis infections.     

Characterization of a 140-kD avian cell surface antigen as a fibronectin-binding molecule

A 140,000-D protein cell surface antigen (140k) complex has been implicated in fibronectin-mediated cell-substratum attachment. We have used three different experimental systems to evaluate the hypothesis that this 140k complex can function as a fibronectin receptor. A monoclonal antibody that binds to the 140k complex specifically inhibits the direct binding of 3H-labeled 75,000-D fibronectin cell-binding fragment (f75k) to chicken embryo fibroblasts in suspension. The 140k complex is retarded in its passage through an affinity column consisting of immobilized f75k, and this interaction is specifically inhibited by a synthetic peptide that contains the fibronectin cell-recognition signal sequence. Finally, exogenous purified 140k complex inhibits the attachment and spreading of chicken embryo fibroblasts on fibronectin-coated substrates. Thus, our results indicate that the 140k complex can bind directly to fibronectin and is likely to be a fibronectin receptor for chicken cells.     

Role of curli fimbriae in mediating the cells of enterohaemorrhagic Escherichia coli to attach to abiotic surfaces

AIMS: The objectives of this study were to evaluate the role of curli in assisting the cells of enterohaemorrhagic Escherichia coli (EHEC) in attaching to abiotic surfaces and to determine the influence of cell-surface contact time on the efficiency of the attachment. METHODS AND RESULTS: Three pairs of EHEC cultures, each with a curli-expressing and a noncurli-expressing variant (O111:H- 7-57C+ and O157:H7 5-9C-, O157:H7 5-11C+ and 5-11C-, as well as O103:H2 7-52C+ and 7-52C-), were allowed to interact with polystyrene, glass, stainless steel and rubber surfaces at 28 degrees C for 24 h (short-term attachment) or 7 days (long-term attachment). The quantities of the cells that attached to the surfaces were measured daily in the long-term attachment study, and in 4 h intervals in the short-term attachment study. Quantification of the cells that attached to the surfaces was accomplished with a crystal violet binding assay. The results of the long-term attachment study indicated that 7-57C+ attached to the polystyrene and glass surfaces more efficiently (P < 0.05) than did 5-9C-. The curli-expressing variant of 5-11 possessed a better ability to adhere to the polystyrene and glass surfaces than did its noncurli-expressing counterpart (P < 0.05). The differences in attachment between 7-52C+ and 7-52C- on polystyrene and stainless steel surfaces were statistically significant (P < 0.05). However, the attachment of the pair on the glass surfaces was statistically insignificant (P > 0.05). In addition, the two members of all three EHEC pairs attached equally well to rubber surfaces (P > 0.05). In the short-term attachment study, only the pair of 7-52 attached differently on glass and stainless steel surfaces (P < 0.05). CONCLUSIONS: These results suggest that curli could be an important cell surface component to mediate the attachment of some EHEC cells to certain abiotic surfaces. Cell-surface contact time could have a significant influence on EHEC attachment to abiotic surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The study signifies a possible role of curli in assisting the cells of EHEC in attaching to food-contact surfaces. It underlines the importance of cleaning and sanitizing food-contact surfaces regularly and thoroughly, and of identifying chemical agents that can effectively remove the attached EHEC cells from these surfaces.     

Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis

Abstract The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier ‘culture and spot’ test. Children (age 4–13; n = 303) and patients ( n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P ⪡ 0.0001). In young children (age 4–5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.     

The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.     

Respiratory syncytial virus promotes Moraxella catarrhalis-induced ascending experimental otitis media

Otitis media (OM) is a polymicrobial disease wherein prior or concurrent infection with an upper respiratory tract virus plays an essential role, predisposing the middle ear to bacterial invasion. In episodes of acute bacterial OM, respiratory syncytial virus (RSV) is the most commonly isolated virus and thus serves as an important co-pathogen. Of the predominant bacterial agents of OM, the pathogenesis of disease due to Moraxella catarrhalis is the least well understood. Rigorous study of M. catarrhalis in the context of OM has been significantly hindered by lack of an animal model. To bridge this gap, we assessed whether co-infection of chinchillas with M. catarrhalis and RSV would facilitate ascension of M. catarrhalis from the nasopharynx into the middle ear. Chinchillas were challenged intranasally with M. catarrhalis followed 48 hours later by intranasal challenge with RSV. Within 7 days, 100% of nasopharynges were colonized with M. catarrhalis and homogenates of middle ear mucosa were also culture-positive. Moreover, within the middle ear space, the mucosa exhibited hemorrhagic foci, and a small volume of serosanguinous effusion was present in one of six ears. To improve upon this model, and based on epidemiologic data, nontypeable Haemophilus influenzae (NTHI) was included as an additional bacterial co-pathogen via intranasal administration four days before M. catarrhalis challenge. With this latter protocol, M. catarrhalis was cultured from the nasopharynx and middle ear homogenates of a maximum of 88% and 79% animals, respectively, for up to 17 days after intranasal challenge with M. catarrhalis. Additionally, hemorrhagic foci were observed in 79% of middle ears upon sacrifice. Thus, these data demonstrated that co-infection with RSV and NTHI predisposed to M. catarrhalis-induced ascending experimental OM. This model can be used both in studies of pathogenesis as well as to investigate strategies to prevent or treat OM due to M. catarrhalis.     

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

Influence of the aglycone region of the substrate binding cleft of Pseudomonas xylanase 10A on catalysis.

Pseudomonas cellulosa xylanase 10A (Pc Xyn10A) contains an extended substrate binding cleft comprising three glycone (-1 to -3) and four aglycone (+1 to +4) subsites and, typical of retaining glycoside hydrolases, exhibits transglycosylation activity at elevated substrate concentrations. In a previous study [Charnock, S. J., et al. (1997) J. Biol. Chem. 272, 2942-2951], it was demonstrated that the -2 subsite mutations E43A and N44A caused a 100-fold reduction in activity against xylooligosaccharides, but did not influence xylanase activity. This led to the proposal that the low activity of these mutants against xylooligosaccharides was due to nonproductive complex formation between these small substrates and the extended aglycone region of the active site. To test this hypothesis, key residues at the +2 (Asn182), +3 (Tyr255), and +4 (Tyr220) subsites were substituted for alanine, and the activity of the mutants against polysaccharides and oligosaccharides was evaluated. All the aglycone mutants exhibited greatly reduced or no transglycosylating activity, and the triple mutants, E43A/Y220A/Y255A and E43A/N182A/Y255A, had activity against xylotriose similar to that of E43A. The aglycone mutations caused an increase in both k(cat) and K(m) against xylan, with N182A/Y220A/Y255A and N182A/Y255A exhibiting 25- and 15-fold higher k(cat) values, respectively, than wild-type Pc Xyn10A. These data indicate that Glu43 plays a role in binding xylooligosaccharides, but not xylan, suggesting that the mechanisms by which Pc Xyn10A binds polysaccharides and oligosaccharides are distinct. The increased k(cat) of the mutants against xylan indicates that the aglycone region of wild-type Pc Xyn10A restricts the rate of catalysis by limiting diffusion of the cleaved substrate, generated at the completion of the k(2) step, out of the active site.     

Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

Process of Infection with Bacteriophage φX174: XXXIV. Kinetics of the Attachment and Eclipse Steps of the Infection

The products of phiX cistrons II, III, and VII are demonstrated to affect the attachment of the phage to its host Escherichia coli C; therefore, by inference, these cistrons influence, directly or indirectly, the structure of proteins in the virus particle. Two of the mutations which alter attachment kinetics, ts79 in cistron III and h in cistron VII, also affect the electrophoretic mobility of the virus and emphasize the role of charge in the attachment interaction with the host. The kinetics for attached phage to go into "eclipse" are first-order and biphasic; about 85% of the phage eclipse at one rate (k(e) = 0.86 min(-1)) and the remainder do so at a distinctly lower rate (k(e) = 0.21 min(-1)). No phiX cistrons yet identified affect the eclipse process. The lowest temperature at which eclipse is detected is 19 C. The Arrhenius activation energy for phage eclipse has the high value of 36.6 kcal/mole, indicating the cooperative nature of the event.     

The role of Branhamella catarrhalis in the "bloody-nose syndrome" of cynomolgus macaques.

During a 15-month period, 25 cynomolgus macaques (Macaca fascicularis) at the Johns Hopkins University were observed to have nasal discharge. Fifteen (60%) of these animals had positive nasal cultures for Branhamella catarrhalis. Clinical signs associated with infection by this bacterium were sneezing, epistaxis, and mucohemorrhagic nasal discharge. Treatment with antibiotics resulted in prompt resolution of clinical signs. Post-therapeutic nasal cultures were negative for B. catarrhalis. Two groups of clinically normal, culture-negative, cynomolgus macaques were inoculated with natural isolates of B. catarrhalis which had been passaged in culture for various amounts of time. Five of the eight animals inoculated became culture-positive and had mild nasal discharge. Presence of blood on nasal swabs was indicative of infection with B. catarrhalis. Three of the inoculated animals had post-swabbing epistaxis. This report documents the role of B. catarrhalis as an upper respiratory pathogen in the cynomolgus monkey which causes mild self-limiting disease reminiscent of the so-called "Bloody-Nose Syndrome." In addition to the obvious clinical significance of this finding to primate clinicians, development of an animal model for human disease caused by this organism may be possible.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Specific features of humoral immunity in cryptogenic fibrosing alveolitis patients

Cryptogenic fibrosing alveolitis (CFA) is a severe autoimmune disease of unclear etiology and prognostically unfavorable. The complexity of the diagnostics of this disease makes it necessary to search for new methods; for this reason immunity in CFA patients must be studied. The study of humoral organ-specific, organ-unspecific and antibacterial immunity of CFA patients revealed that the latter differed from the members of the groups used for comparison by a higher frequency of positive reactions in EIA determinations of IgG antibodies to cytokeratin-8 and Moraxella catarrhalis antigens. In addition, only in CFA patients a high degree of correlation (r=0.88) between these results was established. This made it possible to propose to use these reactions for confirming the diagnosis of CFA and suggested the probable role of M. catarrhalis in triggering autoimmune reactions characteristic of this disease.     

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.