Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes

Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.     

A common pathway for O‐linked protein‐glycosylation and synthesis of capsule in Acinetobacter baumannii

Multi‐drug resistant strains of Acinetobacter baumannii are increasingly being isolated in hospitals worldwide. Among the virulence factors identified in this bacterium there is a general O‐glycosylation system that appears to be important for biofilm formation and virulence, and the capsular polysaccharide, which is essential for resistance to complement killing. In this work, we identified a locus that is responsible for the synthesis of the O‐pentasaccharide found on the glycoproteins. Besides the enzymes required for the assembly of the glycan, additional proteins typically involved in polymerization and transport of capsule were identified within or adjacently to the locus. Mutagenesis of PglC, the initiating glycosyltransferase prevented the synthesis of both glycoproteins and capsule, resulting in abnormal biofilm structures and attenuated virulence in mice. These results, together with the structural analysis of A. baumannii 17978 capsular polysaccharide via NMR, demonstrated that the pentasaccharides that decorate the glycoproteins are also the building blocks for capsule biosynthesis. Two linked subunits, but not longer glycan chains, were detected on proteins via MS. The discovery of a bifurcated pathway for O‐glycosylation and capsule synthesis not only provides insight into the biology of A. baumannii but also identifies potential novel candidates for intervention against this emerging pathogen.     

Phenotypic and genetic differences between opaque and translucent colonies of Vibrio alginolyticus

Many pathogens undergo phase variation between rugose and smooth colony morphology or between opaque and translucent colony morphology, which is mainly due to the variation in the surface polysaccharides. In this study, Vibrio alginolyticus ZJ-51 displayed phase variation between opaque, rugose colonies (Op) and translucent, smooth colonies (Tr). Unlike the vibrios reported previously, Tr cells of ZJ-51 enhanced biofilm formation and motility, but they did not differ from Op cells in the quantity of surface polysaccharides produced. Real time PCR was used to analyze the expression of the genes involved in polysaccharide biosynthesis, flagellar synthesis, and the AI-2 quorum-sensing system. The results revealed that the K-antigen capsule gene cluster (which consists of homologs to the cpsA-K in Vibrio parahaemolyticus) and O-antigen polysaccharide gene cluster (which contains homologs to the wza-wzb-wzc) were significantly more transcribed in Tr cells. The AI-2 quorum-sensing genes showed enhanced expression in the Tr variant which also exhibited greater expression of genes associated with polar flagellar biosynthesis. These results suggest that colony phase variation might affect the virulence and survival ability in the stressful environment inhabited by V. alginolyticus.     

Capsule phase variation in Neisseria meningitidis serogroup B by slipped-strand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion and the outbreak of meningococcal disease

A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule-deficient variants indicated that the capsular polysaccharide was re-expressed at a frequency of 10^?3. Measurement of enzymatic activities involved in the biosynthesis of the α-2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule-negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)₇ residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood-culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo-(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the out-break of meningococcal disease.     

Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production

Summary Transposon and deletion analysis of the cloned K1 capsule biosynthesis genes of Escherichia coli revealed that approximately 17 kb of DNA, split into three functional regions, is required for capsule production. One block (region 1) is required for translocation of polysaccharide to the cell surface and mutations in this region result in the intracellular appearance of polymer indistinguishable on immunoelectrophoresis to that found on the surface of K1 encapsulated bacteria. This material was released from the cell by osmotic shock indicating that the polysaccharide was probably present in the periplasmic space. Insertions in a second block (region 2) completely abolished polymer production and this second region is believed to encode the enzymes for the biosynthesis and polymerisation of the K1 antigen. Addition of exogenous N-acetylneuraminic acid to one insertion mutant in this region restored its ability to express surface polymer as judged by K1 phage sensitivity. This insertion probably defines genes involved in biosynthesis of N-acetylneuraminic acid. Insertions in a third block (region 3) result in the intracellular appearance of polysaccharide with a very low electrophoretic mobility. The presence of the cloned K1 capsule biosynthesis genes on a multicopy plasmid in an E. coli K-12 strain did not increase the yields of capsular polysaccharide produced compared to the K1⁺ isolate from which the genes were cloned.     

Production of glycoprotein vaccines in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.     

Validity and reliability of two brief physical activity questionnaires among Spanish-speaking individuals of Mexican descent

Background

Klebsiella pneumoniae is a leading cause of hospital-acquired urinary tract infections and pneumonia worldwide, and is responsible for many cases of pyogenic liver abscess among diabetic patients in Asia. A defining characteristic of this pathogen is the presence of a thick, exterior capsule that has been reported to play a role in biofilm formation and to protect the organism from threats such antibiotics and host immune challenge.

Findings

We constructed two knockout mutants of K. pneumoniae to investigate how perturbations to capsule biosynthesis alter the cellular phenotype. In the first mutant, we deleted the entire gene cluster responsible for biosynthesis of the extracellular polysaccharide capsule. In the second mutant, we deleted the capsule export subsystem within this cluster. We find that both knockout mutants have lower amounts of capsule but produce greater amounts of biofilm. Moreover, one of the two mutants abolishes fimbriae expression as well.

Conclusions

These results are expected to provide insight into the interaction between capsule biosynthesis, biofilm formation, and fimbriae expression in this organism.     

Mucosal reactive oxygen species decrease virulence by disrupting Campylobacter jejuni phosphotyrosine signaling

Reactive oxygen species (ROS) play key roles in mucosal defense, yet how they are induced and the consequences for pathogens are unclear. We report that ROS generated by epithelial NADPH oxidases (Nox1/Duox2) during Campylobacter jejuni infection impair bacterial capsule formation and virulence by altering bacterial signal transduction. Upon C. jejuni invasion, ROS released from the intestinal mucosa inhibit the bacterial phosphotyrosine network that is regulated by the outer-membrane tyrosine kinase Cjtk (Cj1170/OMP50). ROS-mediated Cjtk inactivation results in an overall decrease in the phosphorylation of C. jejuni outer-membrane/periplasmic proteins, including UDP-GlcNAc/Glc 4-epimerase (Gne), an enzyme required for N-glycosylation and capsule formation. Cjtk positively regulates Gne by phosphorylating an active site tyrosine, while loss of Cjtk or ROS treatment inhibits Gne activity, causing altered polysaccharide synthesis. Thus, epithelial NADPH oxidases are an early antibacterial defense system in the intestinal mucosa that modifies virulence by disrupting bacterial signaling.     

Capsular and extracellular polysaccharides of the diazotrophic rhizobacterium <Emphasis Type="Italic">Herbaspirillum seropedicae</Emphasis> Z78

The diazotrophic endophyte Herbaspirillum seropedicae Z78 was shown to possess a capsule containing two high-molecular-weight glycolipids, one of which was of a lipopolysaccharide nature. These glycolipids differed considerably in the fatty acid composition of their lipid components. The polysaccharide moiety of these glycans was composed of glucose, galactose, glucosamine, galactosamine, and a noncarbohydrate component, butanetetraol. In the culture liquid of H. seropedicae Z78, an extracellular polysaccharide and an extracellular form of lipopolysaccharide were revealed. Fatty acid composition of the extracellular lipopolysaccharide differed from that of the capsular glycoconjugates; the polysaccharide moiety of exoglycans contained only neutral sugars (mannose, glucose, and galactose) and a tetraatomic alcohol, butanetetraol. It is assumed that structural diversity of polysaccharide-containing polymers at the surface of H. seropedicae Z78 cells is conditioned by their different roles in plant colonization and formation of efficient symbiosis.     

Genomic Analysis Reveals the Molecular Basis for Capsule Loss in the Group B Streptococcus Population

The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl trasferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.     

How does Cryptococcus get its coat?

During the past few decades, increasing attention has focused on pathogenic fungi both as fascinating research subjects and as the agents of serious illness in diverse patient populations. In particular, opportunistic fungi such as Cryptococcus neoformans command notice as the ranks of their immunocompromised victims grow. C. neoformans is unique among fungal pathogens for its major virulence factor, a complex polysaccharide capsule. In this article, our current understanding of the structure and biosynthesis of the capsule is reviewed, as are the many questions that remain to be answered about how Cryptococcus gets its coat.     

Synthesis of capsular polysaccharide at the division septum of Streptococcus pneumoniae is dependent on a bacterial tyrosine kinase

One of the main virulence factors of the pathogenic bacterium Streptococcus pneumoniae is the capsule, present at the bacterial surface, surrounding the entire cell. Virtually all the 90 different capsular serotypes of S. pneumoniae, which vary in their chemical composition, express two conserved proteins, Wzd and Wze, which regulate the rate of the synthesis of capsule. In this work, we show that Wzd, a membrane protein, and Wze, a cytoplasmic tyrosine kinase, localize at the bacterial division septum, when expressed together in pneumococcal cells, without requiring the presence of additional proteins encoded in the capsule operon. The interaction between the two proteins and their consequent septal localization was dependent on a functional ATP binding domain of Wze. In the absence of either Wzd or Wze, capsule was still produced, linked to the cell surface, but it was absent from the division septum. We propose that Wzd and Wze are spatial regulators of capsular polysaccharide synthesis and, in the presence of ATP, localize at the division site, ensuring that capsule is produced in co‐ordination with cell wall synthesis, resulting in full encapsulation of the pneumococcal cells.     

Deciphering the role of the capsule of Klebsiella pneumoniae during pathogenesis: A cautionary tale

Extracellular capsule polysaccharides increase the cellular fitness under abiotic stresses and during competition with other bacteria. They are best-known for their role in virulence, particularly in human hosts. Specifically, capsules facilitate tissue invasion by enhancing bacterial evasion from phagocytosis and protect cells from biocidal molecules. Klebsiella pneumoniae is a worrisome nosocomial pathogen with few known virulence factors, but the most important one is its capsule. In this issue, Tan et al. assess the fitness advantage of the capsule by competing a wild-type strain against four different mutants where capsule production is interrupted at different stages of the biosynthetic pathway. Strikingly, not all mutants provide a fitness advantage. They suggest that some mutants have secondary defects altering virulence-associated phenotypes and blurring the role of the capsule in pathogenesis. This study indicates that the K1 capsule in K. pneumoniae is not required for gut colonization but that it is critical for bloodstream dissemination to other organs. These results contribute to clarify the contradictory literature on the role of the Klebsiella capsule during infection. Finally, the varying fitness effects of different capsule mutations observed for K. pneumoniae K1 might apply also to other capsulated diderm bacteria that are facultative or emerging pathogens.     

Ultrastructure of the capsule of Klebsiella pneumoniae and slime of Enterobacter aerogenes revealed by freeze etching

Summary The capsule of Klebsiella pneumoniae type I and slime of Enterobacter aerogenes strain A3 (SL) was examined by electron microscopy using the freeze etch technique. The capsules of K. pneumoniae were found to be composed of several layers of polysaccharide 10 nm thick; while the polysaccharide slime of E. aerogenes strain A3 (SL) was found to be composed of a diffuse network of fibrils. This work represents the first effort to visualize the replica of the unfixed, partially hydrated bacterial capsule or slime in the electron microscope. The slime of E. aerogenes strain A3 (SL) which was purified, and then freeze etched, resembled the layered structure of the capsule of K. pneumoniae. It is suggested that the charge or dielectric constant of the slime polysaccharide polymers was altered during purification, thereby permitting the layering to occur.Paper presented at the Annual Meeting of the American Society for Microbiology, Philadelphia, Pa. (U.S.A.), 1972.     

Role of extracellular matrix components in the formation of biofilms and their contribution to the biocontrol activity of Pseudomonas chlororaphis PCL1606

Pseudomonas chlororaphis PCL1606 (PcPCL1606) displays plant-colonizing features and exhibits antagonistic traits against soil-borne phytopathogenic fungi. Biofilm formation could be relevant for the PcPCL1606 lifestyle, and in this study the role of some putative extracellular matrix components (EMC; Fap-like fibre, alginate and Psl-like polysaccharides) in the biofilm architecture and biocontrol activity of this bacterium were determined. EMC such as the Fap-like fibre and alginate polysaccharide play secondary roles in biofilm formation in PcPCL1606, because they are not fundamental to its biofilm architecture in flow cell chamber, but synergistically they have shown to favour bacterial competition during biofilm formation. Conversely, studies on Psl-like polysaccharide have revealed that it may contain mannose, and that it is strongly involved in the PcPCL1606 biofilm architecture and niche competition. Furthermore, the Fap-like fibre and Psl-like exopolysaccharide play roles in early surface attachment and contribute to biocontrol activity against the white root rot disease caused by Rosellinia necatrix in avocado plants. These results constitute the first report regarding the study of the extracellular matrix of the PcPCL1606 strain and highlight the importance of a putative Fap-like fibre and Psl-like exopolysaccharide produced by PcPCL1606 in the biofilm formation process and interactions with the host plant root.     

Characterization of Regulatory Pathways in Xylella fastidiosa: Genes and Phenotypes Controlled by algU

Many virulence genes in plant bacterial pathogens are coordinately regulated by “global” regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.     

铜绿假单胞菌生物被膜组成及其受群体感应系统和c-di-GMP调控的研究进展

作为人类条件性感染的前三大病原菌之一的铜绿假单胞菌,是一种革兰氏阴性细菌,对免疫功能低下和囊性纤维化患者可以造成严重和持续性感染。造成这种持续感染的原因主要是由于细菌接收外界信号后,在自身调控网络的协同作用下,会依附于固体表面,并产生胞外多糖、基质蛋白和胞外DNA等大分子物质形成高度结构化的膜状复合物将自身包裹形成生物被膜群体结构。生物被膜可以有效帮助细菌定殖、提高细菌对抗菌物质和宿主免疫反应的抵抗能力、促进群落细菌的细胞-细胞之间的信号交流等,是临床治疗中病原菌慢性感染和反复感染最重要的原因之一。本篇综述重点介绍了铜绿假单胞菌生物被膜的各组成成分及其在生物被膜形成中的重要功能,并进一步阐述了群体感应系统(las、rhl、pqs与iqs)和c-di-GMP对铜绿假单胞菌生物被膜形成的调控作用。通过本篇综述可以更清晰地了解细菌生物被膜形成和调控的过程,为开发新的治疗生物被膜感染策略提供帮助。     

Immunochemical characterization of polysaccharide antigens from six clinical strains of Enterococci

Background  

Enterococci have become major nosocomial pathogens due to their intrinsic and acquired resistance to a broad spectrum of antibiotics. Their increasing drug resistance prompts us to search for prominent antigens to develop vaccines against enterococci. Given the success of polysaccharide-based vaccines against various bacterial pathogens, we isolated and characterized the immunochemical properties of polysaccharide antigens from five strains of Enterococcus faecalis and one strain of vancomycin-resistant E. faecium.