Structures of three different neutral polysaccharides of Acinetobacter baumannii, NIPH190, NIPH201, and NIPH615, assigned to K30, K45, and K48 capsule types, respectively, based on capsule biosynthesis gene clusters

Neutral capsular polysaccharides (CPSs) were isolated from Acinetobacter baumannii NIPH190, NIPH201, and NIPH615. The CPSs were found to contain common monosaccharides only and to be branched with a side-chain 1→3-linked β-d-glucopyranose residue. Structures of the oligosaccharide repeat units (K units) of the CPSs were elucidated by 1D and 2D 1H and 13C NMR spectroscopy. Novel CPS biosynthesis gene clusters, designated KL30, KL45, and KL48, were found at the K locus in the genome sequences of NIPH190, NIPH201, and NIPH615, respectively. The genetic content of each gene cluster correlated with the structure of the CPS unit established, and therefore, the capsular types of the strains studied were designated as K30, K45, and K48, respectively. The initiating sugar of each K unit was predicted, and glycosyltransferases encoded by each gene cluster were assigned to the formation of the linkages between sugars in the corresponding K unit.

Bacterial extracellular polysaccharides

Extracellular polysaccharides are as structurally and functionally diverse as the bacteria that synthesise them. They can be present in many forms, including cell-bound capsular polysaccharides, unbound "slime", and as O-antigen component of lipopolysaccharide, with an equally wide range of biological functions. These include resistance to desiccation, protection against nonspecific and specific host immunity, and adherence. Unsurprisingly then, much effort has been made to catalogue the enormous structural complexity of the extracellular polysaccharides made possible by the wide assortment of available monosaccharide combinations, non-carbohydrate residues, and linkage types, and to elucidate their biosynthesis and export. In addition, the work is driven by the commercial potential of these microbial substances in food, pharmaceutics and biomedical industries. Most recently, bacteria-mediated environmental restoration and bioleaching have been attracting much attention owing to their potential to remediate environmental effluents produced by the mining and metallurgy industries. In spite of technological advances in chemistry, molecular biology and imaging techniques that allowed for considerable expansion of knowledge pertaining to the bacterial surface polysaccharides, current understanding of the mechanisms of synthesis and regulation of extracellular polysaccharides is yet to fully explain their structural intricacy and functional variability.

The immunogenicity of chemically derived zwitterionic polysaccharides

Bacterial capsular polysaccharides (PS) which naturally contain zwitterionic charge motifs (ZPS) possess specific immunostimulatory activity, leading to direct activation of antigen-presenting cells (APCs) through Toll-like receptor 2 (TLR2) and of T cells in co-culture systems. When administered intraperitoneally, ZPS and bacteria expressing them are involved in the induction or regulation of T-cell dependent inflammatory processes such as intra-abdominal abscess formation. Moreover it has been published that ZPSs are processed to low molecular weight carbohydrates and presented to T cells through a pathway similar to that used for protein antigens. These findings were in contrast with the paradigm according to which polysaccharides are T-independent antigens unable to be presented in association with MHC class II molecules and unable to induce a protective immune response. For this reason in glycoconjugate vaccines polysaccharides often need to be conjugated to a carrier protein to induce protection. The aim of our work was to generate vaccine candidates with antigen and adjuvant properties in one molecule by the chemical introduction of a positive charge into naturally anionic PS from group B streptococcus (GBS). The resulting zwitterionic PS (ZPS) has the ability to activate human and mouse APCs, and in mixed co-cultures of monocytes and T cells, ZPS induce MHC II-dependent T-cell proliferation and up-regulation of activation markers. TLR2 transfectants show reporter gene transcription upon incubation with ZPS and these stimulatory qualities can be blocked by anti-TLR2 mAbs or by the destruction of the zwitterionic motif. However, in vivo, ZPS used alone as vaccine antigen failed to induce protection against GBS challenge, a result which does not confirm the above mentioned postulate that ZPS are T-cell dependent Ags by virtue of their charge motif. Thus to make ZPS visible to the immune system we have conjugated ZPS with a carrier protein. ZPS-glycoconjugates induce higher T cell and Ab responses to carrier and PS, respectively, compared to control PS-glycoconjugates made with the native polysaccharide form. Moreover, protection of mothers or neonate offspring from lethal GBS challenge is better when mothers are immunized with ZPS-conjugates compared to immunization with PS-conjugates. In TLR2 knockout mice, ZPS-conjugates lose both their increased immunogenicity and protective effect after vaccination. When ZPS are co-administered as adjuvants with unconjugated tetanus toxoid (TT), they have the ability to increase the TT-specific antibody titer. In conclusion, glycoconjugates containing ZPS are potent vaccines. They target Ag to TLR2-expressing APCs and activate these APCs, leading to better T cell priming and ultimately to higher protective Ab titers. Thus, rational chemical design can generate potent novel PS-adjuvants with wide application, including glycoconjugates and co-administration with unrelated protein Ags.

Promiscuity, pheromones and pathogenicity : why all Enterococci are not created equal

Enterococcus faecalis is a Gram-positive, coccus shaped, lactic acid bacterium, with demonstrated ubiquity across multiple anatomical sites. Enterococcus faecalis isolates have been isolated from clinical samples as the etiological agent in patients with overt infections, and from body sites previously thought to be sterile but absent of signs and symptoms of infection. E. faecalis is implicated in both human health and disease, recognized as a commensal, a probiotic and an opportunistic multiply resistant pathogen. E. faecalis has emerged as a key pathogen in nosocomial infections. E. faecalis is well equipped to avert recognition by host cell immune mediators. Antigenic cell wall components including lipotechoic acids are concealed from immune detection by capsular polysaccharides produced by some strains. Thereby preventing complement activation, the pro-inflammatory response, opsonisation and phagocytosis. E. faecalis also produces a suite of enzymes including gelatinase and cytolysin, which aid in both virulence and host immune evasion. The ability of enterococci to form biofilms in vivo further increases virulence, whilst simultaneously preventing detection by host cells. E. faecalis exhibits high levels of both intrinsic and acquired antimicrobial resistance. The mobility of the E. faecalis genome is a significant contributor to antimicrobial resistance, with this species also transferring resistance to other Gram-positive bacteria. Whilst E. faecalis is of increasing concern in nosocomial infections, its role as a member of the endogenous microbiota cannot be underestimated. As a commensal and probiotic, E. faecalis plays an integral role in modulating the immune response, and in providing endogenous antimicrobial activity to enhance exclusion or inhibition of opportunistic pathogens in certain anatomical niches. In this chapter we will review possible mediators of enterococcal transition from commensal microbe to opportunistic pathogen, considering isolates obtained from patients diagnosed with pathogenic infections and those obtained from asymptomatic patients.

Étude des gènes d'Actinobacillus pleuropneumoniae exprimés en conditions d'infection

Actinobacillus pleuropneumoniae est l’agent étiologique de la pleuropneumonie porcine. La bactérie se transmet par voies aériennes et contacts directs. Plusieurs facteurs de virulence ont été identifiés, nommément les polysaccharides capsulaires, les lipopolysaccharide, les exotoxines ApxI à IV et de nombreux mécanismes d’acquisition du fer. Aucun vaccin efficace contre tous les sérotypes de la bactérie n’a encore été élaboré. Afin de mieux comprendre de quelle façon A. pleuropneumoniae régule la transcription de ses nombreux facteurs de virulence et de découvrir de nouvelles cibles potentielles pour l’élaboration de vaccins efficaces, le profil transcriptomique de la bactérie a été étudié dans des conditions simulant l’infection ainsi qu’à la suite d’une infection naturelle aiguë chez l’animal. Des biopuces de première et de seconde génération (AppChip1 et AppChip2) comportant respectivement 2025 cadres de lecture ouverts (ORF) de la version préliminaire du génome d’A. pleuropneumoniae sérotype 5b souche L20 et 2033 ORF de la version finale annotée du même génome ont été utilisées. Dans un premier temps, des expériences réalisées dans des conditions de concentration restreinte en fer ont permis d’identifier 210 gènes différentiellement exprimés, dont 92 étaient surexprimés. Plusieurs nouveaux mécanismes d’acquisition du fer ont pu être identifiés, incluant un système homologue au système YfeABCD de Yersinia pestis, impliqué dans l’acquisition du fer chélaté, ainsi que des gènes homologues aux composantes du système HmbR de Neisseria meningitidis impliqué dans l’acquisition du fer à partir de l’hémoglobine. Dans des conditions de culture permettant la formation de biofilms, les gènes tadC et tadD d’un opéron tad (« tight adherence locus ») putatif, les gènes pgaBC impliqués dans la synthèse d’un polysaccharide de la matrice du biofilm ainsi que deux gènes présentant de fortes homologies avec un gène codant pour l’adhésine auto-transporteur Hsf retrouvée chez Haemophilus influenzae ont montré une surexpression significative. Plusieurs de ces gènes ont également été retrouvés lors d’expériences réalisées avec des cellules épithéliales d’origine pulmonaire en culture, qui ont permis d’identifier 170 gènes différentiellement exprimés après la croissance planctonique au-dessus des cellules, et 131 autres suite à l’adhésion à ces cellules. Parmis les gènes surexprimés, les gènes tadB et rcpA de l’opéron tad putatif, les gènes pgaBC ainsi que le gène codant pour l’homologue d’Hsf ont été retrouvés. En présence de liquide de lavage broncho-alvéolaire (BALF), 156 gènes ont montré un profil d’expression modifié, et le gène apxIVA, identifié comme étant surexprimé, a pu être détecté pour la première fois dans des conditions de croissance in vitro. Finalement, des expériences visant à déterminer les gènes utilisés directement chez l’animal en phase aiguë de la pleuropneumonie porcine ont permis d’identifier 150 gènes qui étaient différentiellement exprimés. En plus d’identifier des gènes d’un possible opéron codant pour un fimbriae de type IV, 3 des 72 gènes surexprimés sont conservés chez tous les sérotypes d’A. pleuropneumoniae et codent pour des protéines ou lipoprotéines de surface. Nos expériences ont permis d’identifier plusieurs nouveaux facteurs de virulence potentiels chez A. pleuropneumoniae ainsi que plusieurs nouvelles cibles potentielles pour l’élaboration de vaccins efficaces contre tous les sérotypes.

Avaliação do potencial biotecnológico de linhagens mutantes de Rhizobium tropici

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Analysis of the memory B cell response against glycoconjugate vaccines

The development of vaccines directed against polysaccharide capsules of S. pneumoniae, H. influenzae and N. meningitidis have been of great importance in preventing potentially fatal infections. Bacterial capsular polysaccharides are T-cell-independent antigens that induce specific antibody response characterized by IgM immunoglobulins, with a very low IgG class switched response and lack of capability of inducing a booster response. The inability of pure polysaccharides to induce sustained immune responses has required the development of vaccines containing polysaccharides conjugated to a carrier protein, with the aim to generate T cell help. It is clear that the immunogenicity of glycoconjugate vaccines can vary depending on different factors, e.g. chemical nature of the linked polysaccharide, carrier protein, age of the target population, adjuvant used. The present study analyzes the memory B cell (MBC) response to the polysaccharide and to the carrier protein following vaccination with a glycoconjugate vaccine for the prevention of Group B streptococcus (GBS) infection. Not much is known about the role of adjuvants in the development of immunological memory raised against GBS polysaccharides, as well as about the influence of having a pre-existing immunity against the carrier protein on the B cell response raised against the polysaccharide component of the vaccine. We demonstrate in the mouse model that adjuvants can increase the antibody and memory B cell response to the carrier protein and to the conjugated polysaccharide. We also demonstrate that a pre-existing immunity to the carrier protein favors the development of the antibody and memory B cell response to subsequent vaccinations with a glycoconjugate, even in absence of adjuvants. These data provide a useful insight for a better understanding of the mechanism of action of this class of vaccines and for designing the best vaccine that could result in a productive and long lasting memory response.

Intravenous immunoglobulin contains a broad repertoire of anticarbohydrate antibodies that is not restricted to the IgG2 subclass

BACKGROUND: Specificities for carbohydrate IgG antibodies, thought to be predominantly of the IgG2 subclass, have never been broadly examined in healthy human subjects. OBJECTIVE: To examine commercial intravenous immunoglobulin (IVIG) preparations for their ability to recognize a wide range of glycans and to determine the contribution of IgG2 to the binding pattern observed. METHODS: We used a glycan microarray to evaluate IVIG preparations and a control mix of similar proportions of human myeloma IgG1 and IgG2 for binding to 377 glycans, courtesy of the Consortium for Functional Glycomics Core H. Glycans recognized were categorized using public databases for their likely cellular sources. IgG2 was depleted from IVIG by using immunoaffinity chromatography, and depletion was confirmed by using nephelometry and surface plasmon resonance. RESULTS: Nearly half of the glycans bound IgG. Some of the glycans with the greatest antibody binding can be found in structures of human pathogenic bacteria (eg, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholera) and nonpathogenic bacteria, including LPS and lipoteichoic acid, capsular polysaccharides, and exopolysaccharides. Surprisingly, depletion of IgG2 had only a modest effect on anticarbohydrate recognition patterns compared with the starting IVIG preparation. Little to no binding activity was detected to human endogenous glycans, including tumor-associated antigens. CONCLUSIONS: This novel, comprehensive analysis provides evidence that IVIG contains a much wider range than previously appreciated of anticarbohydrate IgG antibodies, including those recognizing both pathogenic and non-pathogen-associated prokaryotic glycans.

Effet de Streptococcus Suis sur la capacité de présentation antigénique de cellules dendritiques

Streptococcus suis est un important pathogène porcin et humain, causant méningites et septicémies. Des études suggèrent que S. suis dispose de facteurs de virulence, notamment sa capsule polysaccharidique (CPS), qui lui permettent de moduler les fonctions des cellules dendritiques (DCs), situées à l’interface entre l’immunité innée et adaptative. Les difficultés à développer un vaccin efficace suggèrent aussi une altération de la voie T dépendante. L’objectif général du projet était d’évaluer l’effet de S. suis sur l’activation des cellules T CD4+ ainsi que sur la capacité de présentation antigénique des DCs. Nous avons étudié dans un modèle murin in vivo la réponse T CD4+ mémoire lors d’infections primaire et secondaire. Une faible réponse mémoire centrale a été obtenue, suggérant que la réponse adaptative générée contre S. suis est limitée. Étant donné l’importance du complexe majeur d’histocompatibilité (MHC) de classe II dans la présentation antigénique, nous avons évalué in vitro et in vivo l’expression de ces molécules chez les DCs. Une modulation de l’expression du MHC-II par S. suis a été observée. L’analyse de la transcription de gènes impliqués dans la régulation transcriptionnelle et post-transcriptionnelle du MHC-II nous permet de suggérer que S. suis régule à la baisse la synthèse de nouvelles molécules et favorise leur dégradation lysosomale. Cette stratégie, dans laquelle la CPS ne jouerait qu’un rôle partiel, permettrait à S. suis d’échapper à la réponse adaptative T dépendante. Les résultats de cette étude fourniront de nouvelles perspectives dans la compréhension de la réponse adaptative lors de l’infection par S. suis.

Effet de Streptococcus Suis sur la capacité de présentation antigénique de cellules dendritiques

Streptococcus suis est un important pathogène porcin et humain, causant méningites et septicémies. Des études suggèrent que S. suis dispose de facteurs de virulence, notamment sa capsule polysaccharidique (CPS), qui lui permettent de moduler les fonctions des cellules dendritiques (DCs), situées à l’interface entre l’immunité innée et adaptative. Les difficultés à développer un vaccin efficace suggèrent aussi une altération de la voie T dépendante. L’objectif général du projet était d’évaluer l’effet de S. suis sur l’activation des cellules T CD4+ ainsi que sur la capacité de présentation antigénique des DCs. Nous avons étudié dans un modèle murin in vivo la réponse T CD4+ mémoire lors d’infections primaire et secondaire. Une faible réponse mémoire centrale a été obtenue, suggérant que la réponse adaptative générée contre S. suis est limitée. Étant donné l’importance du complexe majeur d’histocompatibilité (MHC) de classe II dans la présentation antigénique, nous avons évalué in vitro et in vivo l’expression de ces molécules chez les DCs. Une modulation de l’expression du MHC-II par S. suis a été observée. L’analyse de la transcription de gènes impliqués dans la régulation transcriptionnelle et post-transcriptionnelle du MHC-II nous permet de suggérer que S. suis régule à la baisse la synthèse de nouvelles molécules et favorise leur dégradation lysosomale. Cette stratégie, dans laquelle la CPS ne jouerait qu’un rôle partiel, permettrait à S. suis d’échapper à la réponse adaptative T dépendante. Les résultats de cette étude fourniront de nouvelles perspectives dans la compréhension de la réponse adaptative lors de l’infection par S. suis.

Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

Synthesis, structure and conformation of oligo- and polysaccharides

Carbohydrates are a complex group of biomolecules with a high structural diversity. Their almost omnipresent occurrence has generated a broad field of research in both biology and chemistry. This thesis focuses on three different aspects of carbohydrate chemistry, synthesis, structure elucidation and the conformational analysis of carbohydrates. The first paper describes the synthesis of a penta- and a tetrasaccharide related to the highly branched capsular polysaccharide from Streptococcus pneumoniae type 37. In the second paper, the structure of the O-antigenic repeating unit from the lipopolysaccharide of E. coli 396/C1 was determined along with indications of the structure of the biological repeating unit. In addition, its structural and immunological relationship with E. coli O126 is discussed. In the third paper, partially protected galactopyranosides were examined to clarify the origin of an intriguing 4JHO,H coupling, where a W-mediated coupling pathway was found to operate. In the fourth paper, the conformation of methyl a-cellobioside is studied with a combination of molecular dynamics simulations and NMR spectroscopy. In addition to the expected syn-conformation, detection and quantification of anti-ø and anti-ψ conformers was also possible.

Prostate gland and extra-capsular tissue 3D reconstruction and measurement

Currently there are little objective parameters that can quantify the success of one form of prostate surgical removal over another. Accordingly, at Old Dominion University (ODU) we have been developing a process resulting in the use of software algorithms to assess the coverage and depth of extra-capsular soft tissue removed with the prostate by the various surgical approaches. Parameters such as the percent of capsule that is bare of soft tissue and where present the depth and extent of coverage have been assessed. First, visualization methods and tools are developed for images of prostate slices that are provided to ODU by the Pathology Department at Eastern Virginia Medical School (EVMS). The visualization tools interpolate and present 3D models of the prostates. Measurement algorithms are then applied to determine statistics about extra-capsular tissue coverage. This paper addresses the modeling, visualization, and analysis of prostate gland tissue to aid in quantifying prostate surgery success. Particular attention is directed towards the accuracy of these measurements and is addressed in the analysis discussions.