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Background and purpose Nox4 is the major isoform of NADPH oxidase found in the kidney and contributes to the pathogenesis of diabetic nephropathy. However, the molecular mechanisms of increased Nox4 expression induced by hyperglycaemia remain to be elucidated. Here, the role of the connexin32-Nox4 signalling axis in diabetic nephropathy and its related mechanisms were investigated. Experimental approach Diabetes was induced in mice by low-dose streptozotocin (STZ) combined with a high-fat diet. Effects of connexin32 on Nox4 expression and on renal function and fibrosis in STZ-induced diabetic mice were investigated using adenovirus-overexpressing connexin32 and connexin32-deficient mice. Interactions between connexin32 and Nox4 were analysed by co-immunoprecipitation and immunofluorescence assays. Key results Connexin32 was down-regulated in the kidneys of STZ-induced diabetic mice. Overexpression of connexin32 reduced expression of Nox4 and improved renal function and fibrosis in diabetic mice, whereas connexin32 deficiency had opposite effects. Down-regulation of fibronectin expression by connexin32 was not dependent on gap junctional intercellular communication involving connexin32. Connexin32 interacted with Nox4 and reduced the generation of hydrogen peroxide, leading to the down-regulation of fibronectin expression. Mechanistically, connexin32 decreased Nox4 expression by promoting its K48-linked polyubiquitination. Interestingly, Smurf1 overexpression inhibited K48-linked polyubiquitination of Nox4. Furthermore, connexin32 interacted with Smurf1 and inhibited its expression. Conclusion and implications Connexin32 ameliorated renal fibrosis in diabetic mice by promoting K48-linked Nox4 polyubiquitination and degradation via inhibition of Smurf1 expression. Targeting the connexin32-Nox4 signalling axis may contribute to the development of novel treatments for diabetic nephropathy.

Group B Streptococcus serotypes Ia and Ib capsular polysaccharides are key targets for vaccine development. In spite of their immunospecifity these polysaccharides share high structural similarity. Both are composed of the same monosaccharide residues and differ only in the connection of the Neu5Acα2‐3Gal side chain to the GlcNAc unit, which is a β1‐4 linkage in serotype Ia and a β1‐3 linkage in serotype Ib. The development of efficient regioselective routes for GlcNAcβ1‐3[Glcβ1‐4]Gal synthons is described, which give access to different group B Streptococcus (GBS) Ia and Ib repeating unit frameshifts. These glycans were used to probe the conformation and molecular dynamics of the two polysaccharides, highlighting the different presentation of the protruding Neu5Acα2‐3Gal moieties on the polysaccharide backbones and a higher flexibility of Ib polymer relative to Ia, which can impact epitope exposure., Glycosylation: A panel of GBS serotype Ia and Ib fragments was prepared by combined regioselective‐stereoselective approaches and used to unravel polysaccharide conformations responsible for immune specificity.

Porphyromonas gingivalis (Pg) capsule enables evasion from phagocytosis, invasion of keratinocytes, and bacterial survival. In mixed infection, the capsule also participates in coaggregation, which may lead to characteristic virulence not present in the monoinfection. The aim of this study is to evaluate the role of Pg capsule as a virulence factor in coaggregated mixed infection with Fusobacterium nucleatum (Fn).Mixed infections containing Fn and non-capsulated or capsulated strains of Pg were compared with the same infection with lactose as coaggregation inhibitor. Murine experimental periodontitis was used to assess disease severity. Primary polymorphonuclear leukocytes and keratinocytes were used to examine phagocytosis and bacterial invasion, respectively.Mixed infection with capsulated Pg augmented alveolar bone loss compared with that of mixed infection with non-capsulated Pg. Addition of lactose led to attenuation of bone loss in the capsulated mixed infection and to intensification of bone loss in the non-capsulated mixed infection. In the latter mixed infection, Fn evaded phagocytosis, whereas in the capsulated mixed infection Pg displayed a greater capacity for invasion of keratinocytes.Pg capsule was found to serve as a unique virulence factor in mixed infection with Fn. Capsule-dependent coaggregation led to augmented invasion of Pg and may be responsible for the severity of disease after mixed infection with Fn.

IgM molecules circulate in serum as large polymers, mainly pentamers, which can be transported by the poly-Ig receptor (pIgR) across epithelial cells to mucosal surfaces and released as secretory IgM (SIgM). The mucosal SIgM molecules have non-covalently attached secretory component (SC), which is the extracellular part of pIgR which is cleaved from the epithelial cell membrane. Serum IgM antibodies do not contain SC, and have previously been shown to make a conformational change from “a star” to a “staple” conformation upon reaction with antigens on a cell surface, enabling them to activate complement. However, it is not clear if SIgM similarly can induce complement activation. In order to clarify this issue, we constructed recombinant chimeric (mouse/human) IgM antibodies against hapten 5-iodo-4-hydroxy-3-nitro-phenacetyl (NIP) and in addition studied polyclonal IgM formed after immunization with a meningococcal group B vaccine. The monoclonal and polyclonal IgM molecules were purified by affinity chromatography on a column containing human SC in order to isolate joining-chain (J-chain) containing IgM, followed by addition of excess amounts of soluble SC to create SIgM (IgM J+ SC+). These SIgM preparations were tested for complement activation ability and shown to be nearly as active as the parental IgM J+ molecules. Thus, SIgM may offer protection against pathogens at mucosal surface by complement-mediated cell lysis or by phagocytosis mediated by complement receptors present on effector cells on mucosa. This article is protected by copyright. All rights reserved.

Streptococcus agalactiae (Group B Streptococcus, GBS) is associated with diverse diseases in aquatic animals. The capsule polysaccharide (CPS) encoded by the cps gene cluster is the major virulence factor of S. agalactiae; however, limited information is available regarding the pathogenic role of the CPS of serotype Ia piscine GBS strains in fish. Here, a non-encapsulated mutant (Δcps) was constructed by insertional mutagenesis of the cps gene cluster. Mutant pathogenicity was evaluated in vitro based on the killing of whole blood from tilapia, in vivo infections, measuring mutant survival in tilapia spleen tissues and pathological analysis. Compared to wild-type (WT) GBS strain, the Δcps mutant had lower resistance to fresh tilapia whole blood in vitro (p < 0.01), and more easily cleared in tilapia spleen tissue, and was highly attenuated in tilapia and zebrafish. Additionally, compared to the Δcps mutant, numerous GBS strains and severe tissue necrosis were observed in the tilapia spleen tissue infected with WT strains. These results indicated that the CPS is essential for GBS pathogenicity and may serve as a target for attenuation in vaccine development. Gaining a better understanding of the role, the GBS pathogenicity in fish will provide insight into related pathogenesis and host-pathogen interactions.

Analyses of mutations in genes coding for subunits of RNA polymerase always throw more light on the intricate events that regulate the expression of gene(s). Lon protease of Escherichia coli is implicated in the turnover of RcsA (positive regulator of genes involved in capsular polysaccharide synthesis) and SulA (cell division inhibitor induced upon DNA damage). Failure to degrade RcsA and SulA makes lon mutant cells to overproduce capsular polysaccharides and to become sensitive to DNA damaging agents. Earlier reports on suppressors for these characteristic lon phenotypes related the role of cochaperon DnaJ and tmRNA. Here, we report the isolation and characterization of two novel mutations in rpoB gene capable of modulating the expression of cps genes in Δlon strains of E. coli in concert with HNS. clpA, clpB, clpY, and clpQ mutations do not affect this capsule expression suppressor (Ces) phenotype. These mutant RNA polymerases affect rcsA transcription, but per se are not defective either at rcsA or at cps promoters. The results combined with bioinformatics analyses indicate that the weaker interaction between the enzyme and DNA::RNA hybrid during transcription might play a vital role in the lower level expression of rcsA. These results might have relevance to pathogenesis in related bacteria.

Streptococcus mitis is a colonizer of the oral cavity and the nasopharynx, and is closely related to Streptococcus pneumoniae. Both species occur in encapsulated and unencapsulated forms, but in S. mitis the role of the capsule in host interactions is mostly unknown. Therefore, the aim of this study was to examine how capsule expression in S. mitis can modulate interactions with the host with relevance for colonization. The S. mitis type strain, as well as two mutants of the type strain, an isogenic capsule deletion mutant, and a capsule switch mutant expressing the serotype 4 capsule of S. pneumoniae TIGR4, were used. Wild-type and capsule deletion strains of S. pneumoniae TIGR4 were included for comparison. We found that capsule production in S. mitis reduced adhesion to oral and lung epithelial cells. Further, exposure of oral epithelial cells to encapsulated S. mitis resulted in higher interleukin-6 and CXCL-8 transcription levels relative to the unencapsulated mutant. Capsule expression in S. mitis increased the sensitivity to human neutrophil peptide 1-3 but reduced the sensitivity to human β-defensin-3 and cathelicidin. This was in contrast with S. pneumoniae in which capsule expression has been generally associated with increased sensitivity to human antimicrobial peptides (AMPs). Collectively, these findings indicate that capsule expression in S. mitis is important in modulating interactions with epithelial cells, and is associated with increased or reduced susceptibility to AMPs depending on the nature of the AMP.

Objective To determine the prevalence of the different capsular polysaccharide (CP) and major surface-associated non-CP antigen 336 (SP-336) types among Staphylococcus aureus isolated from bovine mastitis cases in Australia and India. Methods A total of 414 strains (154 from Australia, 260 from India) isolated from clinical bovine mastitis were included in the study. Mouse antisera raised against CP types (CP1, CP2, CP5, and CP8) or SP-336 were used in slide agglutination tests and compared with detection of cap1, cap5 and cap8 gene fragments by PCR. Results Serological studies revealed the presence of CP2, CP5, CP8 and SP-336 in 9.1%, 23.4%, 31.8%, and 5.8% of the Australian versus 0.8%, 46.9%, 13.1% and 0% of the Indian isolates, respectively. By PCR, CP1, CP5 and CP8 accounted for 0%, 26.6% and 32.4% of the Australian versus 3.9%, 85% and 8.1% of the Indian isolates, respectively. Conclusions Both PCR and the serological method demonstrated that CP5 and CP8 are the predominant capsular types in Australia, whereas CP5 is the predominant capsular type in India. The study also demonstrated a strong correlation between both methods of typing for CP1, CP5, CP8 and non-typeable S. aureus strains. High-percentage prevalence of non-typeable isolates in both the countries highlights the importance of continued investigations of the identification of unique surface-associated polysaccharide antigens prevalent among S. aureus isolates for the formulation of CP- and SP-based vaccines for bovine mastitis.

Escherichia coli K4 produces a capsule with a chemical structure that resembles chondroitin, a molecule with established chondro protective properties. The endogenous genes pgm and galU are involved in the biosynthesis of UDP-glucose which is a critical intermediate in carbohydrate metabolism and biochemical precursor of UDP-glucuronic acid. Together with UDP-N-acetylgalactosamine, UDP-glucuronic acid is used as sugar donor for capsule biosynthesis. The aim of the study was to evaluate how a change in the pathways leading to UDP-glucuronic acid biosynthesis affected capsular polysaccharide production. One additional copy of pgm and galU was introduced in E. coli K4 and in the previously described recombinant strain EcK4r3. A microbioreactor was used to analyse strain performance with parallel batch experiments, demonstrating increased polysaccharide concentrations and providing data that are comparable to those obtained in larger fermenters. Further experiments on a glutamine enriched medium showed an additional 45% increase of capsule production, maybe indicating the need to balance both branches leading to polymer biosynthesis in order to maximize yields. In the effort towards the establishment of a feasible bio-chondroitin production process this study provides information on how the availability of sugar precursors impacts polysaccharide biosynthesis in E. coli K4, a complex unexplored aspect of a multifaceted process.

Interspecies communication between Porphyromonas gingivalis and Streptococcus gordonii underlies the development of synergistic dual species communities. Contact with S. gordonii initiates signal transduction within P. gingivalis that is based on protein tyrosine (de)phosphorylation. In this study, we characterize a bacterial tyrosine (BY) kinase (designated Ptk1) of P. gingivalis and demonstrate its involvement in interspecies signaling. Ptk1 can utilize ATP for autophosphorylation and is dephosphorylated by the P. gingivalis tyrosine phosphatase, Ltp1. Community development with S. gordonii is severely abrogated in a ptk1 mutant of P. gingivalis, indicating that tyrosine kinase activity is required for maximal polymicrobial synergy. Ptk1 controls the levels of the transcriptional regulator CdhR and the fimbrial adhesin Mfa1 which mediates binding to S. gordonii. The ptk1 gene is in an operon with two genes involved in exopolysaccharide synthesis, and similar to other BY kinases, Ptk1 is necessary for exopolysaccharide production in P. gingivalis. Ptk1 can phosphorylate the capsule related proteins PGN_0224, a UDP-acetyl-mannosamine dehydrogenase, and PGN_0613, a UDP-glucose dehydrogenase, in P. gingivalis. Knockout of ptk1 in an encapsulated strain of P. gingivalis resulted in loss of capsule production. Collectively these results demonstrate that the P. gingivalis Ptk1 BY kinase regulates interspecies communication and controls heterotypic community development with S. gordonii through adjusting the levels of the Mfa1 adhesin and exopolysaccharide.

Given that a significant proportion of children with acute lymphoblastic leukaemia (ALL) lose immune protection to tetanus, diphtheria, and poliomyelitis, revaccination is indicated after chemotherapy. Our randomized pilot study comparing different revaccination schedules suggests that children with ALL might be revaccinated with non-live vaccines as early as 3 months after chemotherapy.

Zwitterionic polysaccharides (ZPSs) from Bacteroides fragilis and Streptococcus pneumoniae display unique T-cell activities. The first synthesis of a hexasaccharide representing two repeating units of the zwitterionic capsular polysaccharide from S. pneumoniae type 1 (Sp1) is reported. Key elements of the approach are stereoselective construction of 1,4-cis-alpha-galactose linkages based on a reactive trichloroacetimidate donor that incorporates a 6-O-acetyl group, which may contribute to the high alpha selectivity in glycosylation. After assembly of the fully protected hexasaccharide from five monosaccharide synthons 2-4, 24 and 25, selective deprotection of the primary hydroxyl groups of the four galactose residues followed by oxidation to the corresponding uronic acids provides hexasaccharide 19. The trisaccharide counterpart 1 was synthesized in similar fashion from three synthons, 2-4. This approach employed both conventional and dehydrative glycosylation methodologies and avoids the use of poorly reactive uronic acid derived glycosyl donors and acceptors.

Eskola J (National Institute for Health and Welfare, Helsinki). Foresight in medicine: current challenges with Haemophilus influenzae type b conjugate vaccines (Foresight). J Intern Med 2010; 267: 241–250. Abstract. An effective vaccine to prevent invasive infections caused by Haemophilus influenzae type b (Hib) bacteria has been available for more than 20 years. Hib conjugate vaccine is safe, efficacious and easy to use, and its cost-benefit ratio is high both in industrialized as well as in developing countries. In spite of this, WHO estimates that every year approximately 8 million children contract life-threatening Haemophilus infections, especially meningitis or severe pneumonia. If we want to take seriously the Millenium Development Goal of reducing the mortality of under 5-year-old children by two-thirds before the year 2015, an effective means to contribute to this would be more efficient use of Hib vaccines [1].

The effect of several cultivation conditions on the kinetics of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14 was studied. The presence in the supernatant of serotype-specific CPS (capsular polysaccharide) during growth was followed by size-exclusion HPLC and, in parallel, confirmed by using a specific latex reagent. The agitation level did not affect the production behaviour, whereas pH maintenance above 6 strongly enhanced both growth and CPS production throughout the cultivation period in flasks. Production of high-molecular-mass polysaccharide was found to be maximal between 5 and 6 h of cultivation, at the end of the exponential phase. By laser light scattering, 90% of this purified CPS product showed a Mw (molecular mass) range from 350 to 1500 kDa, with an average Mw of 921 kDa. Extending the culture to 24 h gave rise to a clear shift of the Mw distribution of the polysaccharide to values lower than 100 kDa. These findings may have strong implications for the large-scale manufacture of the polysaccharide and the associated conjugate vaccine. Fil: Massaldi, Hugo Alberto. Universidad de la Republica Instituto de Higiene; Uruguay. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bessio, M. Inés. Universidad de la Republica Instituto de Higiene; Uruguay Fil: Suárez, Norma. Universidad de la Republica Instituto de Higiene; Uruguay Fil: Texeira, Esther. Universidad de la Republica Instituto de Higiene; Uruguay Fil: Rossi, Silvina. Universidad de la Republica Instituto de Higiene; Uruguay Fil: Ferreira, Fernando. Universidad de la Republica Instituto de Higiene; Uruguay

Anti-polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B capsular polysaccharide were obtained from a phage-displayed peptide library expressing dodecameric peptides, using a human monoclonal antibody (Db3G9). A murine monoclonal antibody (206, F-5) against the serotype 9V capsular polysaccharide identified three peptide mimotopes from the dodecameric peptide library and one from a random pentadecameric peptide library. In ELISA, binding of 206, F-5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type-specific pneumococcal polysaccharide. Peptides were conjugated to keyhole limpet haemocyanin and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti-6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7-keyhole limpet hemocyanin or MP13-keyhole limpet hemocyanin conjugate were significantly and specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.

Staphylococcus aureus capsular polysaccharides are believed to play a role in adhesion to surfaces and may contribute to their antimicrobial resistance, thereby increasing the rates and severity of associated infections. The purpose of this study was to compare the adhesiveness of distinct S. aureus capsular polysaccharides to determine whether adhesiveness was a general or specific feature across different S. aureus strains. Atomic force microscopy was used to confirm the presence or absence of capsular polysaccharides and to measure adhesive forces on a noncapsulated, serotype 8, and serotype 2 strain of S. aureus. Serotype 8 displayed a larger range of adhesive forces (1–19 nN) than the noncapsulated (0–4 nN) and serotype 2 (0–4 nN) strain. The majority of adhesive forces for serotype 8 were in the 10–15 nN range. Removal of capsular polysaccharides gave a marked decrease in adhesive forces measured for serotype 8 and, to a lesser extent, a decrease for serotype 2. Noncapsulated, serotype 8, and serotype 2 S. aureus had water contact angles of 23.8 (±8.9), 34.4 (±2.5), and 56.7 (±11.2) degrees (mean ± standard deviation), respectively. For the first time, capsular polysaccharides from serotype 8 (clinically common) and serotype 2 (clinically rare) were demonstrated to have different physical properties, which may account for variations in studies in which clinical isolates are utilized, and the conflict in proposed roles for capsular polysaccharides on S. aureus is explained. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009

PRR recognize conserved structures on pathogenic microbes and are important for the defense against invading microorganisms. However, accumulating evidence indicates that many pathogens have evolved mechanisms to avoid recognition by PRR. One type of PRR is the macrophage scavenger receptor A (SR-A), which has been shown to play an important role in recognition and non-opsonic phagocytosis of pathogenic bacteria. The bacterial ligands for SR-A have been suggested to be LPS or lipoteichoic acid. Here, we use murine bone marrow-derived macrophages to analyze the role of SR-A in non-opsonic phagocytosis of two major Gram-positive pathogens, Streptococcus agalactiae (group B streptococcus; GBS) and Streptococcus pyogenes. We show that the polysaccharide capsule of GBS and the surface M protein of S. pyogenes, two important virulence factors, prevent SR-A-mediated non-opsonic phagocytosis of streptococci. The sialic acid moiety of the GBS capsule was crucial for its ability to prevent recognition by SR-A. Moreover, we show that a ligand on GBS recognized by SR-A in the absence of capsule is the surface lipoprotein Blr. These findings represent the first example of a microbial strategy to prevent recognition by SR-A and suggest that bacterial surface proteins may be of importance as ligands for SR-A.

Neisseria meningitidis type A (MenA) is a Gram-negative encapsulated bacterium that may cause explosive epidemics of meningitis, especially in the sub-Saharan region of Africa. The development and manufacture of an efficient glycoconjugate vaccine against Neisseria meningitidis A is greatly hampered by the poor hydrolytic stability of its capsular polysaccharide, which is made up of (1-->6)-linked 2-acetamido-2-deoxy-alpha-D-mannopyranosyl phosphate repeating units. Since this chemical lability is a product of the inherent instability of the phosphodiester bridges, here we report the synthesis of phosphonoester-linked oligomers of N-acetyl mannosamine as candidates for stabilised analogues of the corresponding phosphate-bridged saccharides. The installation of each interglycosidic phosphonoester linkage was achieved by Mitsunobu coupling of a glycosyl C-phosphonate building block with the 6-OH moiety of a mannosaminyl residue. Each of the synthesised compounds contains an O-linked aminopropyl spacer at its reducing end (alpha- or beta-oriented) to allow for protein conjugation. The relative affinities of the synthetic molecules were investigated by a competitive ELISA assay and showed that a human polyclonal anti-MenA serum can recognise both the phosphonoester-bridged fragments 1-3 and their monomeric subunits, glycosides 20 and 21. Moreover, the biological results suggest that the abilities of these compounds to inhibit the binding of a specific antibody to MenA polysaccharide are dependent on the chain lengths of the molecules, but independent on the orientations of the anomeric linkers.

When protein-polysaccharide conjugated vaccines were first developed for the immunization of small children against meningitis caused by infection with Haemophilus influenzae type b (Hib), the vaccine preparations varied in immunogenicity. Testing for immunogenicity was time-consuming and alternative analytical procedures for determining vaccine quality were unsatisfactory. For example, due to the very high molecular weight of the vaccine particles, immunogens could only be physically characterized as a fraction in the void volume of Sepharose gel filtration. In search of better analytical methods, a computer-assisted electrophoretic technique for analyzing such vaccines was developed in the period from 1983 to 1995. This new approach made it possible to analyze highly negatively charged particles as large as or larger than intact viruses. 2-D gel patterns were generated that varied depending on the conditions of the particular vaccine preparation and were therefore characteristic of each vaccine sample. Thus, vaccine particle populations with a continuous size variation over a wide range (polydisperse) could be characterized according to size and free mobility (related to particle surface net charge density). These advances are reviewed in this article, since the developed methods are still a promising tool for vaccine quality control and for predicting immunogen effectiveness in the production of vaccines. The technique is potentially beneficial for Hib immunogens and other high-molecular-mass vaccines. Additional biomedical applications for this nondenaturing electrophoretic technique are briefly discussed and detailed information about computational and mathematical procedures and theoretical aspects is provided in the Appendices.

Recurrent otitis media are frequently intractable during childhood. It is unclear whether recurrent otitis media is caused by etiological bacteria colonization or by new infections. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were isolated from the nasopharynx of 7 otitis-prone and 2 non-prone children with recurrent otitis media. Plural bacterial species and strains were found in all children while affected by otitis media. The same strain was repeatedly isolated from all otitis-prone children even after administration of antibiotics but was not from the non-prone children. Antibiotic susceptibility did not differ significantly among the same repeatedly isolated strains. This pilot study suggests that the etiological bacteria tend to colonize and is hard to eliminate in otitis-prone children.

Escherichia coli K1 is part of a reservoir of adherent, invasive facultative pathogens responsible for a wide range of human and animal disease including sepsis, meningitis, urinary tract infection and inflammatory bowel syndrome. A prominent virulence factor in these diseases is the polysialic acid capsular polysaccharide (K1 antigen), which is encoded by the kps/neu accretion domain inserted near pheV at 67 map units. Some E. coli K1 strains undergo form (phase) variation involving loss or gain of O-acetyl esters at carbon positions 7 or 9 of the individual sialic acid residues of the polysialic acid chains. Acetylation is catalysed by the receptor-modifying acetyl coenzyme-A-dependent O-acetyltransferase encoded by neuO, a phase variable locus mapping near the integrase gene of the K1-specific prophage, CUS-3, which is inserted in argW at 53.1 map units. As the first E. coli contingency locus shown to operate by a translational switch, further investigation of neuO should provide a better understanding of the invasive K1 pathotype. Minimal estimates of morbidity and economic costs associated with human infections caused by extraintestinal pathogenic E. coli strains such as K1 indicate at least 6.5 million cases with attendant medical costs exceeding 2.5 billion US dollars annually in the United States alone.

Meningitis caused by Escherichia coli K1 is a serious illness in neonates with neurological sequelae in up to 50% of survivors. A high degree of bacteremia is required for E. coli K1 to cross the blood-brain barrier, which suggests that the bacterium must evade the host defence mechanisms and survive in the bloodstream. We previously showed that outer membrane protein A (OmpA) of E. coli binds C4b-binding protein (C4bp), an inhibitor of complement activation via the classical pathway. Nevertheless, the exact mechanism by which E. coli K1 survives in serum remains elusive. Here, we demonstrate that log phase (LP) OmpA+ E. coli K1 avoids serum bactericidal activity more effectively than postexponential phase bacteria. OmpA- E. coli cannot survive in serum grown to either phase. The increased serum resistance of LP OmpA+ E. coli is the result of increased binding of C4bp, with a concomitant decrease in the deposition of C3b and the downstream complement proteins responsible for the formation of the membrane attack complex. C4bp bound to E. coli K1 acts as a cofactor to factor I in the cleavage of both C3b and C4b, which shuts down the ensuing complement cascade. Accordingly, a peptide corresponding to the complement control protein domain 3 of C4bp sequence, was able to compete with C4bp binding to OmpA and cause increased deposition of C3b. Thus, binding of C4bp appears to be responsible for survival of E. coli K1 in human serum.

The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes.

Most Staphylococcus aureus express a serotype 5 or 8 capsular polysaccharide (CP). However, 20-25% of human isolates and up to 86% of bovine strains of S. aureus are non-typeable (NT), i.e. non-reactive with antibodies to CP types 1, 2, 5 or 8. A vaccine that targets the S. aureus CP would not protect against NT strains. The aim of this study was to characterize NT S. aureus isolates at the molecular level to explain their lack of type 5 or 8 capsule production. The cap5(8) locus was present in all 22 NT clinical isolates from humans, eight of 21 bovine isolates, and in all eight sequenced strains. NT strains positive for the cap5(8) transcript had mutations within essential capsule genes and could be complemented in trans. S. aureus strains with reduced cap5(8) transcript had mutations within the cap5A promoter, decreased RNAIII levels, or a truncated arlR gene product. More than one mutation was identified in several isolates. The cap5(8) locus was replaced by IS257 in 13 of 21 NT bovine isolates of S. aureus. Lack of capsule expression in NT S. aureus can be explained by multiple mechanisms, and the data argue against the existence of capsule serotypes other than 1, 2, 5 and 8.

The export of large negatively charged capsular polysaccharides across the outer membrane represents a significant challenge to Gram negative bacteria. In the case of Escherichia coli group 2 capsular polysaccharides, the mechanism of export across the outer membrane was unknown, with no identified candidate outer membrane proteins. In this paper we demonstrate that the KpsD protein, previously believed to be a periplasmic protein, is an outer membrane protein involved in the export of group 2 capsular polysaccharides across the outer membrane. We demonstrate that KpsD and KpsE are located at the poles of the cell and that polysaccharide biosynthesis and export occurs at these polar sites. By in vivo chemical cross-linking and MALDI-TOF-MS analysis we demonstrate the presence of a multi-protein biosynthetic/export complex in which cytoplasmic proteins involved in polysaccharide biosynthesis could be cross-linked to proteins involved in export across the inner and outer membranes. In addition, we show that the RhsA protein, of previously unknown function, could be cross-linked to the complex and that a rhsA mutation reduces K5 biosynthesis suggesting a role for RhsA in coupling biosynthesis and export.

Fourier transform infrared (FTIR) spectroscopy has shown remarkable ability in distinguishing between bacterial species and identifying bacterial colony structures, when used in tandem with methods such as cluster analysis, principal component analysis, or linear discriminant analysis. The present work was aimed to evaluate the potential of FTIR–microscopy (FTIR-MSP) to distinguish between different serotypes and capsular quantities of Streptococcus pneumoniae. In general, the results obtained have consistently proven that the spectral information at the region 900–1185 cm−1 was sufficient to distinguish between various pneumococcal serotypes. Moreover, the method was able to differentiate between S. pneumoniae phase variants on the basis of their relative carbohydrate content. The unsupervised cluster analysis of the samples showed differences, not only in the carbohydrate content, but also in the region 1350–1480 cm−1, which is dominated by absorptions due to lipids and phospholipids. This approach proved to be useful for the distinction between S. pneumoniae serotypes and between phase variants, which were shown to acquire different pathogenic capacity. © 2006 Wiley Periodicals, Inc. Biopolymers 83:434–442, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The capsule of Cryptococcus neoformans can undergo dramatic enlargement, a phenomenon associated with virulence. A prior study that used Ab to the capsule as a marker for older capsular material concluded that capsule growth involved the intermixing of new and old capsular material with displacement of older capsular polysaccharide towards the surface. Here we have revisited that question using complement (C), which binds to capsular polysaccharide covalently, and cannot redistribute by dissociation and binding at different sites. The experimental approach involved binding of C to cells with small capsules, inducing capsule growth, and following the location of C relative to the cell wall as the capsule enlarged. C remained close to the cell wall during capsule growth, indicating that capsule enlargement occurred by addition of new polysaccharide near the capsule edge. This conclusion was confirmed by an independent method that employed radioactive metabolic labelling of newly synthesized capsule with 3H-mannose followed by gradual capsular stripping with gamma-radiation. Capsule growth proceeded to a certain size, which was a function of cell size, and was not degraded when the cells were transferred to a non-inducing medium. During budding, an opening appeared in the capsule of the mother cell that permitted the nascent bud to separate. Scanning EM suggested that a physical separation formed between the capsules of the mother and daughter cells during budding, which may avoid mixture between both capsules. Our results indicate that C. neoformans capsular enlargement also occurs by apical growth and that budding results in capsular rearrangements.

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is a steroid hormone that regulates calcium metabolism. Besides, 1alpha,25(OH)(2)D(3 )also has pronounced immunomodulatory effects: it strongly inhibits dendritic cell (DC) maturation and impairs IL-12 production. We studied the effect of 1alpha,25(OH)(2)D(3 )on the antibody response to pneumococcal capsular polysaccharide (caps-PS) serotype 3. 1alpha,25(OH)(2)D(3) inhibited the IgG2a antibody response to caps-PS serotype 3. Besides, 1alpha,25(OH)(2)D(3) also inhibited IL-12 production and maturation of DC. Anti-IL-12 and exogenous IL-12, respectively, inhibited and stimulated the IgG2a antibody response to caps-PS serotype 3. Exogenous IL-12 abrogated the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3, indicating that the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3 was mediated through IL-12. In conclusion, we demonstrate that 1alpha,25(OH)(2)D(3) has an inhibitory effect on the IgG2a antibody response to caps-PS serotype 3, and that this effect was mediated trough IL-12.

Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon. In the course of this study, it was found that when grown in vitro on tryptic soy agar, A. salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide. A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides. Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose[3-[(N-acetyl-L-alanyl)amido]-3-deoxy-D-quinovose, Qui3NAlaNAc] and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: [-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-]n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group. CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form. Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated.

Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children worldwide. During childhood, acquisition of antibody directed against the polysaccharide capsule of the organism, presumably as a result of asymptomatic carriage, confers protection and disease is much less common after the age of 4 years. Like other polysaccharides, the polyribosyl ribitol phosphate (PRP) of the Hib capsule is a T-independent antigen and not immunogenic when administered as a vaccine in infancy. Because the highest rates of disease occur in the first 2 years of life, efficacious Hib vaccines have been designed by covalently linking the PRP capsule to a carrier protein that recruits T-cell help for the polysaccharide immune response and induces anti-PRP antibody production even in the first 6 months of life. Introduction of Hib protein-polysaccharide conjugate vaccines into many industrialized countries over the past 15 years has resulted in the virtual elimination of invasive Hib disease. However, despite the success of the vaccine programme several factors may interfere with the effectiveness of the vaccine in the routine programme, as observed in the UK recently. Such factors may include interference with other concomitant vaccines, waning immunity in the absence of booster doses of vaccine, and reduced natural boosting as a result of decreased transmission of the organism. However, the burden of disease remains highest in resource-poor countries and urgent efforts are needed to provide the benefits of this vaccine for children living in regions where it cannot be used for economic and logistical reasons.

Protection against infections with Streptococcus pneumoniae is mediated by antibodies against the capsular polysaccharides (caps-PS). Here we show that in in vitro experiments CD4+ T lymphocytes stimulate and CD8+ T lymphocytes inhibit the human anti-caps-PS antibody response. Using antagonistic anti-CD40 and antagonistic anti-CD40 ligand (CD40L) monoclonal antibodies, we showed that the CD4+ T lymphocyte-mediated stimulation is dependent on the CD40-CD40L interaction. The role of CD40L was further illustrated by the observation that CD4+ T lymphocytes obtained from a patient with hyper-IgM syndrome were unable to enhance the immune response to caps-PS. Furthermore, CD4+ T lymphocytes from cord blood, which did not express CD40L in response to stimulation with caps-PS, failed to stimulate the antibody response of adult B lymphocytes to caps-PS. These in vitro findings were confirmed by in vivo experiments in which SCID/SCID mice were reconstituted with human mononuclear cells. Furthermore, we showed that caps-PS induce production of IL-4, IL-6, IL-10, and IFN-gamma, and that this enhanced production was inhibited by blocking the CD40-CD40L interaction. This is the first demonstration that the human immune response to caps-PS, which is markedly regulated by T lymphocytes, is dependent on the CD40-CD40L interaction.

Objectives: To assess the completeness of ACIR data for south-eastern Sydney children at 12 to

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. The two polyanions, K4 and defructosylated K4, are separated and readily determined within 30 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship was found for the two polysaccharides over a wide range of concentrations, from approximately 30 ng (0.5 microg/microL) to 210 ng (3.5 microg/microL). The described method was used to evaluate the defructosylation process of K4 under drastic acid conditions.

The aim of the study was to assess age-specific incidences of community-acquired pneumonia (CAP) caused by Streptococcus pneumoniae and diagnosed serologically in a child population. The study was population-based, and prospective, and performed in primary health care settings. During a surveillance period of 12 months from 1981-1982, all pneumonia cases in a defined child population (57% urban residents) were registered prospectively. In total, 201 CAP cases were diagnosed (mean age 5.6 years; 57% boys; 58% urban residents). S. pneumoniae etiology was studied by antibody and immune complex (IC) assays to C-polysacchride (C-PS), type-specific capsular polysaccharides (CPS), and to pneumolysin (Ply), in acute and convalescent sera. Serologic evidence of S. pneumoniae etiology was indicated in 57(28%) cases, 35(61%) being mixed infections with other microbes. The distribution of pneumococcal cases was 44%, 30% and 26% in the three 5-year age groups, respectively. There were 33 (58%) males and 34 (60%) urban residents. In total, 26 cases were identified by antibody assays and 35 cases by IC assays, 26/35 being positive in acute sera. Responses to C-PS, CPS and Ply, when antibody and IC results are combined, were found equally often in 23-25 cases. The total annual incidence of pediatric S. pneumoniae CAP was 6.4/1000/year. S. pneumoniae etiology was found in 28% of the children and was similar at all ages. The incidence of pneumococcal CAP was assessed for the first time, being high (19/1000/year) in 0- to 4-year-old urban boys and rather stable (5-9/1000/year) in all other groups by age, sex and residence.

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltaHexAFrc-GalNAc for K4 and deltaHexA-GalNAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 micro g for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 micro g for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

The aromatic hydrocarbon-degrading bacterium, Pseudomonas putida G7, produces exopolymers of potential interest in biotechnological applications. These exopolymers have been shown to have significant metal-binding ability. To initiate the study of the metal-polymer interactions, we explored the physical and chemical nature of the P. putida G7 exopolysaccharide, a major component of the exopolymer. A capsular structure was observed by light microscopy surrounding both planktonic and attached cells in biofilms after immunofluorescence staining with polyclonal antiserum raised against planktonic cells. Further work with planktonic cells showed that the immunostained capsule remained associated with young (log phase) cells, whereas older (stationary phase) cells lost their capsular material to the external milieu. Visualization of frozen, hydrated stationary phase cells by cryo-field emission scanning electron microscopy (cryoFESEM) revealed highly preserved extracellular material. In contrast, conventional scanning electron microscopy (SEM) of stationary phase cells showed rope-like material that most probably results from dehydrated and collapsed exopolymer. Both capsular and released exopolymers were separated from cells, and the released extracellular polysaccharide (EPS) was purified. Deoxycholate-polyacrylamide gel electrophoresis (PAGE) and silver/alcian blue staining of the partially purified material showed that it contained both EPS and lipopolysaccharide (LPS). Further purification of the EPS using a differential solubilization technique to remove LPS yielded highly purified EPS. Gas chromatography-mass spectrometry revealed that the purified EPS contained the monosaccharides, glucose, rhamnose, ribose, N-acetylgalactosamine and glucuronic acid. The structural and chemical properties of the P. putida EPS described here increase our understanding of the mechanisms of toxic metal binding by this well-known Proteobacterium.

In this study, we re-investigated the previously characterized RcsC (sensor His-kinase) --RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli. The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus. As judged from the current general view as to the His --Asp phosphorelay, this RcsC --RcsB framework is somewhat puzzling, because RcsC appears to contain both a His-kinase domain and a receiver domain, but not a histidine (His)-containing phosphotransmitter domain (e.g. HPt domain). We thus suspected that an as yet unknown mechanism might be underlying in this particular His --Asp phosphorelay system. Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His-containing phosphotransmitter (named YojN) is essential for this signalling system. A revised model is proposed in which the multistep RcsC --YojN --RcsB phosphorelay is implicated. It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E. coli cells during colony formation on the surface of agar plates, namely swarming.

The human antibody response to the capsular polysaccharide of Haemophilus influenzae type b is predominated by antibodies expressing a light-chain-associated idiotype designated HibId-1. HibId-1 is expressed by kappa light chains encoded by either the A2 or A18 variable region genes. In this report we use site-directed mutagenesis and molecular modeling to show that HibId-1 expression is determined by residues in the first and second complimentarity determining regions that are widely separated in the primary sequence, but closely juxtaposed by the tertiary folding of the mature light chain molecule. Of the known human light chains, only alleles of A2 and A18 encode these residues at these positions in their germline configuration. VIG10, a mouse monoclonal antibody of unknown specificity that expresses HibId-1, and 23F.2, an A2-utilizing Streptococcus pneumoniae 23F polysaccharide-specific human Fab fragment that lacks HibId-1, provide examples of the HibId-1 determinant both arising and being lost by somatic mutation. In addition, we show that the residues responsible for HibId-1 expression can be disassociated from those required for antigen binding.

Strains of Escherichia coli expressing the K1 polysaccharide capsule colonize the large intestine of newborn infants, and are the leading cause of Gram-negative septicaemia and meningitis in the neonatal period. We used signature-tagged mutagenesis (STM) to identify genes that E. coli K1 requires to colonize the gastrointestinal (GI) tract. A total of 2140 mTn5 mutants was screened for their capacity to colonize the GI tract of infant rats, and 16 colonization defective mutants were identified. The mutants have transposon insertions in genes affecting the synthesis of cell surface structures, membrane transporters, transcriptional regulators, enzymes in metabolic pathways, and in genes of unknown function, designated dgc (defective in GI colonization). Three dgcs are absent from the whole genome sequence of E. coli K-12, although related sequences are found in other pathogenic strains of E. coli and in Shigella flexneri. Additionally, immunohistochemistry was used to define the nature of the colonization defect in five mutants including all dgc mutants. STM was successfully applied to examine the factors involved in E. coli K1 colonization, and the findings are relevant to the pathogenesis of other enteric infections.

In this study we examined the ability of capsular polysaccharide type 2 (PS) from Streptococcus pnemoniae to induce secretory and cellular responses in peritoneal macrophages. Tumour cytotoxicity induced by preincubation with PS was demonstrated to be concentration-dependent. PS-induced tumouricidal activity was partially abrogated by anti-tumour necrosis factor (TNF)-alpha and inhibitor of nitric oxide, whereas anti-interferon (IFN)-alpha/beta antibody and the scavengers of reactive oxygen intermediates had no effect. In addition, supernatants from macrophages treated with PS contained TNF-alpha, and their iNOS-enzymatic activity was significantly increased. Thus, the tumouricidal activity induced by PS appeared to be mediated by the production of TNF-alpha and nitrite. Treatment of macrophages with PS increased the expression of CD14, the receptor for lipolysaccharide (LPS)/LPS-binding protein. Moreover, blocking antibody to CD14 abrogated partially TNF-alpha and nitrite induction by PS, suggesting that the PS-induced CD14 upregulation was correlated with secretion of TNF-alpha and nitrite. Taken together, these results demonstrate that PS may induce macrophage-secretory and cellular activities, in part, possibly via CD14-dependent pathway.

Antibodies specific for capsular polysaccharide epitopes mediate immunity to encapsulated bacterial pathogens, and accordingly, vaccine development has focused upon the induction of these specificities. Efficacious vaccines, consisting of either polysaccharide alone or polysaccharide coupled to protein carriers, have been developed for a number of pathogens. Their clinical importance notwithstanding, these vaccines serve as model antigens to study the genetic and somatic forces molding adaptive immunity in man. In this article we review progress aimed at delineating the structure and dynamics of the human antibody repertoire to the Haemophilus influenzae type b polysaccharide (Hib PS), a system which has been studied from infancy to old age. Collectively, the data reveal a repertoire which is encoded by a relatively large number of germline variable (V) region gene segments, but which is typically expressed within individuals as a markedly restricted, oligoclonal population. One particular V domain has attained canonical status because of its high penetrance at the population level and its predominance in individual repertoires. Although this combining site is assembled in early infancy and retains its prominence throughout life, its frequency of expression, affinity and protective function are dictated by the molecular form of the Hib PS immunogen (vaccine). The determinants of Hib PS binding affinity can include both germline and somatically-acquired V region polymorphisms. We discuss how these properties of the Hib PS repertoire could impact immunity to Hib, and we consider the implications of these findings towards understanding the evolution of immunoglobulin germline V genes.

Staphylococcus aureus is the most common cause of septic arthritis. This disease often leads to severe joint destruction and high mortality. An experimental model of S. aureus arthritis has been developed to study the course of inflammation and joint destruction, to elucidate the role of bacterial and host factors for joint pathology and mortality, and to develop therapeutical and preventive devices against septic arthritis and sepsis. Results show that the innate immune system is crucial in defending the host against staphylococcal infection while components of the specific immune system, T and B lymphocytes and their products, are detrimental to the host, mediating joint destruction and increasing mortality rates. Staphylococcal capsule polysaccharides, toxins, cell wall-attached adhesins and possibly also the chromosomal DNA are virulence determinants in S. aureus arthritis. Several vaccine candidates have recently been described which protects against staphylococcal infections, e.g. staphylococcal surface polysaccharides, enterotoxins devoid of their superantigenic properties and collagen adhesin. There are also new approaches suggested for treatment of ongoing infections, such as the combined use of antibiotics and corticosteroids.

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40 degrees C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP-Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43 degrees C (at least 25 times lower than those observed when the growth temperature was 37 degrees C). The synthesis of these enzymes increased rapidly when bacteria grown at 43 degrees C were transferred to 37 degrees C and decreased dramatically when cells grown at 37 degrees C were transferred to 43 degrees C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.

Objective: Few data are available on invasive disease due to Streptococcus pneumoniae in representative Australian childhood populations. This study aimed to determine the age-specific incidence of invasive pneumococcal disease in Sydney children. Methodology: Population-based prospective study where isolates of Streptococcus pneumoniae from normally sterile sites were identified through an established laboratory surveillance network. Isolates came from children aged under 15 years living within the boundaries of Central, Eastern, Southern, Western and South-western Sydney Area Health Services from 1 July 1991 to 30 June 1996. Results: Invasive pneumococcal disease was identified in 320 children during a 5-year period, of whom 193 (60%) were under 2 years of age. The incidence per 100 000 children was 12.7 per 100 000 (95% CI: 11.4–14.2/100 000) under 15 years; 31.7 (95% CI 28.1–35.7) under 5 years, and 45.5 (95% CI 38.9–53.3) under 2 years. The incidence of pneumococcal meningitis in children aged under 2 years was 10.5 per 100 000 (95% CI: 7.4–14.5/100 000). Conclusions: The incidence of childhood invasive pneumococcal disease in Sydney was stable during 1991–96 and comparable to rates reported from other industrialized countries. There was no evidence of any change in pneumococcal disease incidence with reduction in invasive Haemophilus influenzae type b (Hib) disease following introduction of Hib immunization.

Uridine diphosphate galacturonate 4-epimerases Introduction (UDPGLEs) are enzymes that convert UDP-glucuronate into UDP-galacturonate. Although the presence of UDPGLEs hasbeen reported in prokaryotic and eukary-otic organisms, the genes coding for these enzymes are completely unknown. The galacturonic acid-con-taining capsular polysaccharide of Streptococcus pneumoniae type 1 is synthesized through the action of a specific UDPGLE. We have constructed a defined deletion mutant in the cap1J gene (one of the 15 cap1 genes responsible for the synthesis of the type 1 cap-sule) that exhibited an unencapsulated phenotype. This mutant was unable to synthesize UDPGLE, sug-gesting that Cap1J was the type 1-specific UDPGLE of S. pneumoniae. Escherichia coli cells harbouring the recombinant plasmid pRMM38 (cap1J ) overpro-duced a 40 kDa protein, characterized as Cap1J on the basis of the N-terminal amino acid sequence analysis, and expressed high levels of enzymatically active Cap1J epimerase. Cap1J was partially purified, although puri-fication to electrophoretic homogeneity inactivated the enzyme irreversibly. The enzyme has the following characteristics: Km for UDP-glucuronate, 0.24 mM; pH optimum, 7.5; equilibrium constant (in the direction of UDP-galacturonate formation), 1.3; and an approximate Mr of 80 000 for the active form. The Cap1J protein exhibited a fluorescence emission spectrum similar to that of NADH. Upon inactivation with p-hydroxymer-curibenzoate, the addition of NADþ and 2-mercap-toethanol were sufficient to reactivate the enzyme. In an early work, Smith et al. (1957) first reported the isolation of UDP-galacturonic acid (UDP-GalA) from a type 1 Streptococcus pneumoniae isolate. Additional studies showed that cell-free extracts prepared from type 1 pneumococci contained an enzymatic activity capable of converting UDP-glucuronic acid (UDP-GlcA) into UDP-GalA (Smith et al., 1958; 1960). UDP-GalA serves as a pre-cursor for the synthesis of type 1 pneumococcal capsular polysaccharide, as demonstrated by the finding that mutants deficient in UDP-glucuronate 4-epimerase (UDP-GLE) (EC 5.1.3.6) activity were unencapsulated (Austrian et al., 1959). Several independent reports have also detec-ted the presence of UDPGLE in the cyanobacterium Ana-baena flos-aquae (Ankel and Tischer, 1969; Gaunt et al., 1972; 1974; Maitra et al., 1974) and in different plants (Neufeld et al., 1958; Feingold et al., 1960; Neufeld, 1966; Dalessandro and Northcote, 1977). Unfortunately, none of these enzymes have been purified to sufficient homo-geneity, and attempts to clone the corresponding genes have not been documented, hampering a reliable charac-terization of this type of protein. We have reported recently the characterization of the gene cluster responsible for capsular polysaccharide bio-synthesis in type 1 strains of S. pneumoniae (Garcı´a et al., 1997; Mun˜oz et al., 1997) and identified the gene cap1K as that coding for the UDP-glucose (UDP-Glc) dehydrogenase responsible for the synthesis of UDP-GlcA from UDP-Glc. We report here experimental evidence, We thank J. C. Díez Masa for his invaluable help with the chromatographic separation of UDP-glucuronic acid and UDP- galacturonic acid, F. J. Medrano for his advice with the spectro- fluorometer, P. García and J. L. García for critical reading of the manuscript, and E. Cano, M. Carrasco and V. Muñoz for tech- nical assistance. This work was supported by grant PB96-0809 from the Dirección General de Investigación Científica y Téc- nica. R. Muñoz was a recipient of a Contrato Temporal de Investigadores from the CSIC.

The hyaluronic acid capsule of group A Streptococcus (GAS) is an important virulence factor, but little is known about mechanisms that regulate capsule expression. Transposon Tn916 mutagenesis of the poorly encapsulated M-type 3 GAS strain DLS003 produced a transconjugant that exhibited a mucoid colony morphology, reflecting increased hyaluronic acid capsule production. Analysis of chromosomal DNA sequence immediately downstream of the transposon insertion identified two open reading frames, designated csrR and csrS, which exhibited sequence similarity to bacterial two-component regulatory systems. We constructed an in-frame deletion mutation within csrR, which encodes the putative response component. Replacement of the native csrR gene in the DLS003 chromosome with the mutant allele resulted in a sixfold increase in capsule production and a corresponding increase in transcription of the has operon, which contains the essential genes for hyaluronic acid synthesis. Increased capsule production by the csrR mutant strain was associated with enhanced resistance to complement-mediated opsonophagocytic killing in vitro and with a 500-fold increase in virulence in mice. These results establish CsrR as a negative regulator of hyaluronic acid capsule synthesis and suggest that it is part of a two-component regulatory system that influences capsule expression and virulence.

Capsular structures of Porphyromonas gingivalis have been correlated to the pathogenicity in animal models. Six polysaccharide capsular serotypes have recently been described in P. gingivalis. In the present study, virulence of the P. gingivalis strains of the six capsular serotypes was compared with strains of the non-capsular serotype of P. gingivalis in a mouse model. All 18 encapsulated strains caused a spreading type of infection with exudate, often accompanied by ulceration and necrosis of the skin and a significant loss of body weight. All three non-encapsulated strains tested induced localized abscesses, although a spreading type of infection was occasionally observed. Illness of the mice was almost always associated with recovery of encapsulated P. gingivalis from blood, kidneys, lungs and spleens. A statistically significant contrast was found in the average weight change during the experiment between the mice infected with the K- serotype strains and the mice infected with the encapsulated P. gingivalis strains. Differences in severity of illness and the recovery of P. gingivalis from mouse tissues were recorded between strains of the same serotype. The differences in virulence within a capsular serotype suggest that the capsule is an important but not the only determining virulence factor for P. gingivalis.