Characterisation of a capsular polysaccharide from Moraxella nonliquefaciens CCUG 348T.

Moraxella nonliquefaciens is a commensal of the human upper respiratory tract (URT) but on rare occasions is recovered in cases of ocular, septic and pulmonary infections. Hence there is interest in the pathogenic determinants of M. nonliquefaciens, of which outer membrane (OM) structures such as fimbriae and two capsular polysaccharide (CPS) structures, →3)-ß-D-GalpNAc-(1→5)-ß-Kdop-(2→ and →8)-α-NeuAc-(2→, have been reported in the literature. To further characterise its surface virulence factors, we isolated a novel CPS from M. nonliquefaciens type strain CCUG 348T. This structure was elucidated using NMR data obtained from CPS samples that were subjected to various degrees of mild acid hydrolysis. Together with GLC-MS data, the structure was resolved as a linear polymer composed of two GalfNAc residues consecutively added to Kdo, →3)-ß-D-GalfNAc-(1→3)-α-D-GalfNAc-(1→5)-α-(8-OAc)Kdop-(2→. Supporting evidence for this material being CPS was drawn from the proposed CPS biosynthetic locus which encoded a potential GalfNAc transferase, a UDP-GalpNAc mutase for UDP-GalfNAc production and a putative CPS polymerase with predicted GalfNAc and Kdo transferase domains. This study describes a unique CPS composition reported in Moraxella spp. and offers genetic insights into the synthesis and expression of GalfNAc residues, which are rare in bacterial OM glycans.

Otitis media: recent advances in otitis media vaccine development and model systems.

Otitis media is an inflammatory disorder of the middle ear caused by airways-associated bacterial or viral infections. It is one of the most common childhood infections as globally more than 80% of children are diagnosed with acute otitis media by 3 years of age and it is a common reason for doctor's visits, antibiotics prescriptions, and surgery among children. Otitis media is a multifactorial disease with various genetic, immunologic, infectious, and environmental factors predisposing children to develop ear infections. Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the most common culprits responsible for acute otitis media. Despite the massive global disease burden, the pathogenesis of otitis media is still unclear and requires extensive future research. Antibiotics are the preferred treatment to cure middle ear infections, however, the antimicrobial resistance rate of common middle ear pathogens has increased considerably over the years. At present, pneumococcal and influenza vaccines are administered as a preventive measure against otitis media, nevertheless, these vaccines are only beneficial in preventing carriage and/or disease caused by vaccine serotypes. Otitis media caused by non-vaccine serotype pneumococci, non-typeable H. influenza, and M. catarrhalis remain an important healthcare burden. The development of multi-species vaccines is an arduous process but is required to reduce the global burden of this disease. Many novel vaccines against S. pneumoniae, non-typeable H. influenza, and M. catarrhalis are in preclinical trials. It is anticipated that these vaccines will lower the disease burden and provide better protection against otitis media. To study disease pathology the rat, mouse, and chinchilla are commonly used to induce experimental acute otitis media to test new therapeutics, including antibiotics and vaccines. Each of these models has its advantages and disadvantages, yet there is still a need to develop an improved animal model providing a better correlated mechanistic understanding of human middle ear infections, thereby underpinning the development of more effective otitis media therapeutics. This review provides an updated summary of current vaccines against otitis media, various animal models of otitis media, their limitations, and some future insights in this field providing a springboard in the development of new animal models and novel vaccines for otitis media.

Efficacy of an impression disinfectant solution after repeated use: An in vitro study.

Statement of problem: There are very few studies using Benzalkonium Chloride (BAC) as an active disinfection agent for immersion techniques and there are no studies investigating the efficacy of repeated use of a disinfectant solution. Purpose: This study evaluated an impression disinfectant by testing bacterial contamination of disinfectant batches used in a clinical setting after repeated use. Materials and methods: Liquid samples were collected from impression disinfectant solutions used to disinfect dental impressions taken at a university dental clinic. The experimental samples (500 ml from 1 L of solution) were collected from teaching and professional clinics and the in-house commercial processing laboratory and stored at room temperature each day of clinic operation over five weeks. To determine to what extent the disinfectant efficacy of the active product decreased over time, the following tests were carried out: a. Inoculation b. Gram staining technique c. Matrix Assisted Laser Desorption/Ionization Mass spectrometry (MALDI- MS). Microbial growth was monitored and photographed. A culture revival was made from colonies grown on sheep blood agar, to isolate pure colonies incubated for 24 h at 37 °C. Each morphologically distinct type of colony was gram stained and MALDI spectrometry analysis was performed using the VITEK MS (BioMerieux Inc.). Results: Evidence of growth of bacteria was detected in teaching clinics' samples, and no growth from the professional clinic or the commercial laboratory. Conclusions: The study demonstrated that the impression disinfectant solution tested is effective against common oral bacteria, despite some rare species such as Bacillus circulans, Bacillus horneckiae, Bacillus altitudinis/pumilus and Bacillus cereus showing evidence of survival in solutions used for disinfection of impressions. However, in a high use teaching clinic environment its efficacy deteriorated. Though a second level disinfection protocol in the commercial laboratory-maintained impression disinfection.

Moraxella ovis and Moraxella bovoculi lipooligosaccharide biosynthesis genes, and structural characterisation of oligosaccharide from M. ovis 354T.

Moraxella ovis is a Gram-negative bacterium isolated from sheep conjunctivitis cases and is a rare isolate of infectious bovine keratoconjunctivitis (IBK). This species is closely related to M. bovoculi, another species which can also be isolated from IBK, or cattle upper respiratory tract (URT). Prior to molecular identification techniques, M. bovoculi was frequently misclassified as M. ovis. We previously described the structure of two oligosaccharides (lipooligosaccharide-derived, minor and major glycoforms) from M. bovoculi 237T (type strain, also ATCC BAA-1259T). Here, we have identified the genetic loci for lipooligosaccharide synthesis in M. ovis 354T (NCTC11227) and compared it with M. bovoculi 237T. We identified genes encoding the known glycosyltransferases Lgt6 and Lgt3 in M.ovis. These genes are conserved in Moraxella spp., including M bovoculi. We identified three further putative OS biosynthesis genes that are restricted to M. ovis and M. bovoculi. These encode enzymes predicted to function as GDP-mannose synthases, namely a mannosyltransferase and a glycosyltransferase. Adding insight into the genetic relatedness of M.ovis and M. bovoculi, the M. ovis genes have higher similarity to those in M. bovoculi genotype 2 (nasopharyngeal isolates from asymptomatic cattle), than to M. bovoculi genotype 1 (isolates from eyes of IBK-affected cattle). Sequence analysis confirmed that the predicted mannosyltransferase in M. bovoculi 237T is interrupted by a C>T polymorphism. This mutation is not present in other M. bovoculi strains sequenced to date. We isolated and characterised LOS-derived oligosaccharide from M. ovis 354T. GLC-MS and NMR spectroscopy data revealed a heptasaccharide structure with three ß-D-Glcp residues attached as branches to the central 3,4,6-α-D-Glcp, with subsequent attachment to Kdo. This inner core arrangement is consistent with the action of Lgt6 and Lgt3 glycosyltransferases. Two α-D-Manp residues are linearly attached to the 4-linked ß-D-Glcp, consistent with the presence of the two identified glycosyltransferases. This oligosaccharide structure is consistent with the previously reported minor glycoform isolated from M. bovoculi 237T.

Structural Insights into Pixatimod (PG545) Inhibition of Heparanase, a Key Enzyme in Cancer and Viral Infections.

Pixatimod (PG545), a heparan sulfate (HS) mimetic and anticancer agent currently in clinical trials, is a potent inhibitor of heparanase. Heparanase is an endo-ß-glucuronidase that degrades HS in the extracellular matrix and basement membranes and is implicated in numerous pathological processes such as cancer and viral infections, including SARS-CoV-2. To understand how PG545 interacts with heparanase, we firstly carried out a conformational analysis through a combination of NMR experiments and molecular modelling which showed that the reducing end ß-D-glucose residue of PG545 adopts a distorted conformation. This was followed by docking and molecular dynamics simulations to study the interactions of PG545 with heparanase, revealing that PG545 is able to block the active site by binding in different conformations, with the cholestanol side-chain making important hydrophobic interactions. While PG545 blocks its natural substrate HS from binding to the active site, small synthetic heparanase substrates are only partially excluded, and thus pentasaccharide or larger substrates are preferred for assaying this class of inhibitor. This study provides new insights for the design of next-generation heparanase inhibitors and substrates.

Structural characterisation of the oligosaccharide from Moraxella bovoculi type strain 237 (ATCC BAA-1259) lipooligosaccharide.

The Gram-negative bacterium Moraxella bovoculi is associated with infectious bovine keratoconjunctivitis (IBK), colloquially known as 'pink-eye'. IBK is an extremely contagious ocular disease of cattle. We report here the structure of the oligosaccharide derived from the lipooligosaccharide from M. bovoculi type strain 237 (also known as ATCC BAA-1259T). GLC-MS and correlation NMR analysis of the oligosaccharide revealed 5 sugar residues, with a notable central branched 3,4,6-α-D-Glcp. An additional α-D-Manp was present ~30% on the sub-terminal α-D-Manp of the 4-linked branch. This oligosaccharide structure was consistent with other members of the Moraxellaceae where no heptose was present and 5-linked Kdo was directly attached to the central 3,4,6-α-D-Glcp.

Using electronic dental records to establish a surveillance system for dental decay in rural Western Alaska.

OBJECTIVES: Previous surveys have demonstrated high rates of early childhood caries (ECC) in the Alaska Native (AN) population of western Alaska. There are many challenges to providing dental care in this road-less Yukon-Kuskokwim Delta region. The regional Tribal Health Organization implemented an electronic dental record (EDR) system in the late 1990s. We explored use of the EDR to establish an oral health surveillance system in children. METHODS: We contracted with EDR software developers to implement calculation of a summary count of decayed (d), missing (m) or filled (f) primary (dmft) score for each individual. We calculated the yearly average dmft scores for 2011-2019 for children aged 3 and 5 years with a comprehensive exam in a given year. We also assessed the number of children undergoing full mouth dental rehabilitation (FMDR). We used US census data population estimates for these age groups to calculate rates. RESULTS: Over the 9-year period, 2,427 3-year-old children (47 percent of all 3-year olds over this period), received a comprehensive exam; increasing from 24 percent in 2011 to 62 percent in 2019. Their average dmft score over the 9-years was 6.4 with a significant annual decline over this period. Seventy percent of AN children who turned 6 between 2015 and 2019 had received at least one FMDR. CONCLUSIONS: An oral health surveillance system has been established in western Alaska using the Electronic Dental Record. High rates of ECC and FMDR were observed. This surveillance system will allow assessments of ECC prevalence and impact of dental interventions.

Immunological characterisation of truncated lipooligosaccharide-outer membrane protein based conjugate vaccine against Moraxella catarrhalis and nontypeable Haemophilus influenzae.

Moraxella catarrhalis and nontypeable Haemophilus influenzae are important bacterial causes of otitis media in children and respiratory diseases in adults. Lipooligosaccharide (LOS) from M. catarrhalis and outer membrane protein 26 (OMP26) from NTHi are major surface antigens identified as potential vaccine components against these organisms. We previously constructed M. catarrhalis in which LOS is truncated, but contains a structure common to the three known serotypes of M. catarrhalis. OMP26 is known to enhance clearance of NTHi following vaccination in animal models, so was chosen as the carrier protein. In this study, we conjugated wild-type and truncated M. catarrhalis detoxified-LOS to a recombinant modified OMP26, rOMP26VTAL. Vaccination of mice with these conjugates resulted in a significant increase in anti-LOS and anti-rOMP26VTAL IgG levels. Importantly, mouse antisera showed complement-mediated bactericidal activity against all M. catarrhalis serotype A and B strains and a NTHi strain tested. Serotypes A & B make up more than 90% of isolates. These data suggest that the LOS and OMP based conjugate can be used as vaccine components and require further investigation in animal models.

The role of lipooligosaccharide in the biological activity of Moraxella bovis strains Epp63, Mb25 and L183/2, and isolation of capsular polysaccharide from L183/2.

The Gram-negative bovine pathogen Moraxella bovis is a causative agent of Infectious bovine keratoconjunctivitis, 'pink-eye' that affects cattle. Here we report that strain L183/2 has the same capsular polysaccharide (CPS) of unsulfated chondroitin, as does strain Mb25, whereas strain Epp63 does not express CPS. NMR analysis of the oligosaccharides (OS) derived from the lipooligosaccharides (LOS) in these three strains by NMR has shown that strain Mb25 and Epp63 have the same OS structure with a terminal N-acetylgalactosamine ((1S)-GalaNAc) residue →4,6-linked. Strain L183/2 lacks the (1 S)-GalaNAc residue. The biological role of M. bovis LOS was assessed by comparing the LOS from strains Epp63, Mb25 and L183/2 and truncated Epp63 LOS variants. LOS truncation affected M. bovis growth rate, susceptibility to antibiotics, detergents, bovine serum bactericidal activity, endotoxicity and adherence to HeLa cells.

A direct-sensing galactose chemoreceptor recently evolved in invasive strains of Campylobacter jejuni.

A rare chemotaxis receptor, Tlp11, has been previously identified in invasive strains of Campylobacter jejuni, the most prevalent cause of bacterial gastroenteritis worldwide. Here we use glycan and small-molecule arrays, as well as surface plasmon resonance, to show that Tlp11 specifically interacts with galactose. Tlp11 is required for the chemotactic response of C. jejuni to galactose, as shown using wild type, allelic inactivation and addition mutants. The inactivated mutant displays reduced virulence in vivo, in a model of chicken colonization. The Tlp11 sensory domain represents the first known sugar-binding dCache_1 domain, which is the most abundant family of extracellular sensors in bacteria. The Tlp11 signalling domain interacts with the chemotaxis scaffolding proteins CheV and CheW, and comparative genomic analysis indicates a likely recent evolutionary origin for Tlp11. We propose to rename Tlp11 as CcrG, Campylobacter ChemoReceptor for Galactose.

Identification and characterisation of a biosynthetic locus for Moraxella bovis lipo-oligosaccharide.

Moraxella bovis is a Gram-negative gammaproteobacterium and is one of the causative agents of infectious bovine keratoconjunctivitis. The structure of lipooligosaccharide (LOS) from strain Epp63 was recently elucidated. In the present study a genetic locus of seven encoding genes with high similarity to glycosyltransferases has been identified. Mutation of these putative glycosyltransferase genes resulted in M. bovis mutant bacteria that expressed truncated LOS structures. The structures of the oligosaccharide (OS) expressed by the mutant strains were elucidated and demonstrated the role of the glycosyltransferase enzymes in the LOS biosynthesis of M. bovis. The glycosyltransferase genes designated lgt1, lgt3, and lgt6 are highly similar to the genes in the related bacterium M. catarrhalis. In addition, there are syntenic similarities with the corresponding LOS biosynthesis locus in M. catarrhalis and other members of Moraxellaceae.

An Unusual Carbohydrate Conformation is Evident in Moraxella catarrhalis Oligosaccharides.

Oligosaccharide structures derived from the lipooligosaccharide of M. catarrhalis show that the highly branched glucose-rich inner core of the oligosaccharide has an altered conformation compared to the most truncated tetra-glucose-Kdo lgt1/4Δ oligosaccharide structure. Addition of one residue each to the (1-4) and (1-6) chains to give the lgt2Δ oligosaccharide is the minimum requirement for this conformational change to occur. Extensive molecular modeling and NMR investigations have shown that the (1-3), (1-4), and (1-6) glycosidic linkages from the central α-D-Glcp have significantly altered conformational preferences between the two structures. For the lgt1/4Δ oligosaccharide the (1-3) and (1-4) linkage populates predominantly the syn minimum on the conformational free energy map and for the (1-6) linkage conformational flexibility is observed, which is supported by 1H-NMR T1 measurements. For the lgt2Δ oligosaccharide the unusual "(1-4)anti-ψ(1-6)gg" conformation, which could be confirmed by long-range NOE signals, is a dominant conformation in which the oligosaccharide is very compact with the terminal α-D-GlcNAc residue folding back towards the center of the molecule leading to an extensive intra-molecular hydrophobic interaction between the terminal residues. Comparing effective H-H distances, which were calculated for conformational sub-ensembles, with the NOE distances revealed that typically multiple conformations could be present without significantly violating the measured NOE restraints. For lgt2Δ the presence of more than one conformation is supported by the NOE data.

Elucidation of the structure of the oligosaccharide from wild type Moraxella bovis Epp63 lipooligosaccharide.

Moraxella bovis is a Gram-negative microorganism that causes Infectious Bovine Keratoconjunctivitis (IBK), colloquially known as 'Pink eye' in cattle worldwide. Lipopolysaccharides/lipooligosaccharides are the predominant glycans on the surface of Gram-negative microorganisms. Structural elucidation of the oligosaccharide structure of the rough phenotype of Moraxella bovis strain Epp63 was determined using GC-MS, methylation analysis, and NMR spectroscopy. The oligosaccharide is a branched structure that comprises 10 sugars in addition to KDO. The unusual features of this oligosaccharide include the fact that the oligosaccharide is devoid of heptose. The KDO residue is directly attached to a (→4,6)-branched glucose and additionally contains a terminal open chain acetal-linked N-acetylgalactosamine, (1S)-GalaNAc residue →4,6-linked to a sub-terminal galactose residue.

Characterisation of a multi-ligand binding chemoreceptor CcmL (Tlp3) of Campylobacter jejuni.

Campylobacter jejuni is the leading cause of human gastroenteritis worldwide with over 500 million cases annually. Chemotaxis and motility have been identified as important virulence factors associated with C. jejuni colonisation. Group A transducer-like proteins (Tlps) are responsible for sensing the external environment for bacterial movement to or away from a chemical gradient or stimulus. In this study, we have demonstrated Cj1564 (Tlp3) to be a multi-ligand binding chemoreceptor and report direct evidence supporting the involvement of Cj1564 (Tlp3) in the chemotaxis signalling pathway via small molecule arrays, surface plasmon and nuclear magnetic resonance (SPR and NMR) as well as chemotaxis assays of wild type and isogenic mutant strains. A modified nutrient depleted chemotaxis assay was further used to determine positive or negative chemotaxis with specific ligands. Here we demonstrate the ability of Cj1564 to interact with the chemoattractants isoleucine, purine, malic acid and fumaric acid and chemorepellents lysine, glucosamine, succinic acid, arginine and thiamine. An isogenic mutant of cj1564 was shown to have altered phenotypic characteristics of C. jejuni, including loss of curvature in bacterial cell shape, reduced chemotactic motility and an increase in both autoagglutination and biofilm formation. We demonstrate Cj1564 to have a role in invasion as in in vitro assays the tlp3 isogenic mutant has a reduced ability to adhere and invade a cultured epithelial cell line; interestingly however, colonisation ability of avian caeca appears to be unaltered. Additionally, protein-protein interaction studies revealed signal transduction initiation through the scaffolding proteins CheV and CheW in the chemotaxis sensory pathway. This is the first report characterising Cj1564 as a multi-ligand receptor for C. jejuni, we therefore, propose to name this receptor CcmL, Campylobacter chemoreceptor for multiple ligands. In conclusion, this study identifies a novel multifunctional role for the C. jejuni CcmL chemoreceptor and illustrates its involvement in the chemotaxis pathway and subsequent survival of this organism in the host.

1H NMR spectroscopic studies establish that heparanase is a retaining glycosidase.

Heparanase is an endo-ß-glucuronidase that cleaves heparan sulfate side chains of proteoglycans in basement membranes and the extracellular matrix (ECM). Heparanase is implicated in several diverse pathological processes associated with ECM degradation such as metastasis, inflammation and angiogenesis and is thus an important target for anti-cancer and anti-inflammatory drug discovery. Heparanase has been classed as belonging to the clan A glycoside hydrolase family 79 based on sequence analysis, secondary structure predictions and mutagenic analysis, and thus it has been inferred that it is a retaining glycosidase. However, there has been no direct experimental evidence to support this conclusion. Herein we describe (1)H NMR spectroscopic studies of the hydrolysis of the pentasaccharide substrate fondaparinux by heparanase, and provide conclusive evidence that heparanase hydrolyses its substrate with retention of configuration and is thus established as a retaining glycosidase. Knowledge of the mechanism of hydrolysis may have implications for future design of inhibitors for this important drug target.

Investigation into the feasibility of thioditaloside as a novel scaffold for galectin-3-specific inhibitors.

Galectin-3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this ß-galactose-binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin-1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour-challenged mice. A novel approach--replacing galactose with its C2 epimer, talose--offers an alternative framework, as extensions at C2 permit exploitation of a galectin-3-specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin-3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin-3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.

Substrate recognition of a structure motif for phosphorylcholine post-translational modification in Neisseria meningitidis.

Neisseria meningitidis is a human pathogen that can cause life threatening meningitis and sepsis. Pili of Neisseria are one of the major virulence factors in host-pathogen interaction. Pilin of N.meningitidis is post-translationally modified by a glycan and two phosphorylcholines (ChoP). ChoP modifications have been found to have an important role in bacterial colonisation and invasion. Unlike N. gonorrhoeae, ChoP modifications on pili seem to be restricted to the C-terminus of pilin protein in N. meningitidis. In this study, we investigate the substrate recognition of phosphorylcholine transferase. We found that a single sequence of D-A-S after the disulphide bond of pilin protein is able to form a motif for ChoP modifications and the charge residue in this motif and the local structure are essential for the substrate recognition.

Structural heterogeneity of terminal glycans in Campylobacter jejuni lipooligosaccharides.

Lipooligosaccharides of the gastrointestinal pathogen Campylobacter jejuni are regarded as a major virulence factor and are implicated in the production of cross-reactive antibodies against host gangliosides, which leads to the development of autoimmune neuropathies such as Guillain-Barré and Fisher Syndromes. C. jejuni strains are known to produce diverse LOS structures encoded by more than 19 types of LOS biosynthesis clusters. This study demonstrates that the final C. jejuni LOS structure cannot always be predicted from the genetic composition of the LOS biosynthesis cluster, as determined by novel lectin array analysis of the terminal LOS glycans. The differences were shown to be partially facilitated by the differential on/off status of three genes wlaN, cst and cj1144-45. The on/off status of these genes was also analysed in C. jejuni strains grown in vitro and in vivo, isolated directly from the host animal without passaging, using immunoseparation. Importantly, C. jejuni strains 331, 421 and 520 encoding cluster type C were shown to produce different LOS, mimicking asialo GM(1), asialo GM(2) and a heterogeneous mix of gangliosides and other glycoconjugates respectively. In addition, individual C. jejuni colonies were shown to consistently produce heterogeneous LOS structures, irrespective of the cluster type and the status of phase variable genes. Furthermore we describe C. jejuni strains (351 and 375) with LOS clusters that do not match any of the previously described LOS clusters, yet are able to produce LOS with asialo GM(2)-like mimicries. The LOS biosynthesis clusters of these strains are likely to contain genes that code for LOS biosynthesis machinery previously not identified, yet capable of synthesising LOS mimicking gangliosides.

Synthesis of a novel pentasaccharide core component from the lipooligosaccharide of Moraxella catarrhalis.

The novel pentasaccharide [p-(trifluoroacetamido)phenyl]ethyl 3-O-ß-D-glucopyranosyl-4-O-ß-D-glucopyranosyl-6-O-[2-O-(α-D-glucopyranosyl)-ß-D-glucopyranosyl]-α-D-glucopyranoside (1), which includes a linker moiety to enable facile coupling to an antigenic protein, was synthesised as a component of a potential vaccine candidate against the Gram-negative bacterium Moraxella catarrhalis. This microorganism is one of three principal causative agents of otitis media in children. The pentasaccharide represents a common cross-serotype (A, B and C) structure from the lipooligosaccharides of Moraxella catarrhalis.

Three Streptococcus pneumoniae sialidases: three different products.

Streptococcus penumoniae is a major human pathogen responsible for respiratory tract infections, septicemia, and meningitis and continues to produce numerous cases of disease with relatively high mortalities. S. pneumoniae encodes up to three sialidases, NanA, NanB, and NanC, that have been implicated in pathogenesis and are potential drug targets. NanA has been shown to be a promiscuous sialidase, hydrolyzing the removal of Neu5Ac from a variety of glycoconjugates with retention of configuration at the anomeric center, as we confirm by NMR. NanB is an intramolecular trans-sialidase producing 2,7-anhydro-Neu5Ac selectively from α2,3-sialosides. Here, we show that the first product of NanC is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) that can be slowly hydrated by the enzyme to Neu5Ac. We propose that the three pneumococcal sialidases share a common catalytic mechanism up to the final product formation step, and speculate on the roles of the enzymes in the lifecycle of the bacterium.