Structure analysis of the Staphylococcus aureus UDP-N-acetyl-mannosamine dehydrogenase Cap5O involved in capsular polysaccharide biosynthesis

Bacterial UDP-sugar dehydrogenases are part of the biosynthesis pathway of extracellular polysaccharides. These compounds act as important virulence factors by protecting the cell from opsonophagocytosis and complement-mediated killing. In Staphylococcus aureus, the protein Cap5O catalyzes the oxidation of UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid. Cap5O is crucial for the production of serotype 5 capsular polysaccharide that prevents the interaction of bacteria with both phagocytic and nonphagocytic eukaryotic cells. However, details of its catalytic mechanism remain unknown. We thus crystallized Cap5O and solved the first structure of an UDP-N-acetyl-mannosamine dehydrogenase. This study revealed that the catalytic cysteine makes a disulfide bond that has never been observed in other structurally characterized members of the NDP-sugar dehydrogenase family. Biochemical and mutagenesis experiments demonstrated that the formation of this disulfide bridge regulates the activity of Cap5O. We also identified two arginine residues essential for Cap5O activity. Previous data suggested that Cap5O is activated by tyrosine phosphorylation, so we characterized the phosphorylation site and examined the underlying regulatory mechanism.     

Turnover of cell surface-bound capsular polysaccharide in Staphylococcus aureus

The capsular polysaccharide (CPS) of Staphylococcus aureus strain Smith was labelled by growth of bacteria in the presence of radioactive N-acetylglucosamine and was separated from labelled cell wall components by affinity chromatography on wheat germ agglutinin following dissolution of the cells by lysostaphin. The products were partially characterised chemically and immunochemically. Similar labelled components were found in the culture fluid during growth. In a pulse-chase experiment, cell-bound CPS was released continuously into the culture fluid at the same rate as cell wall turnover and there was no evidence of direct excretion of CPS.     

Structure of the type 5 capsular polysaccharide of Staphylococcus aureus

The Staphylococcus aureus type 5 capsular polysaccharide is composed of 2-acetamido-2-deoxy-L-fucose (1 part), 2-acetamido-2-deoxy-D-fucose (1 part), and 2-acetamido-2-deoxy-D-mannuronic acid (1 part). On the basis of methylation analysis, optical rotation, high-field one- and two-dimensional 1H- and 13C-n.m.r. experiments, and selective cleavage with 70% aqueous hydrogen fluoride, the polysaccharide was found to be a partially O-acetylated (50%) polymer of the repeating trisaccharide unit, [----4)-3-O-Ac-beta-D-ManpNAcA-(1----4)-a-L-FucpNAc-(1----3) -beta-D-FucpNAc-(1----]n.     

Identification of a gene essential for O-acetylation of the Staphylococcus aureus type 5 capsular polysaccharide

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a trisaccharide repeating unit of (→ 4)-3-O-Ac-β- D -ManNAcA p -(1 → 4)-α- L -FucNAc p -(1 → 3)-β- D -FucNAc p -(1→). Tn 918 mutagenesis of strain Reynolds yielded a mutant that produced wild-type levels of O-deacetylated CP5. The site and orientation of the single transposon insertion in mutant JL232 were determined by analysis of Southern blots and amplification of DNA flanking the transposon. DNA sequencing revealed that Tn 918 was inserted within an open reading frame of 627 bp. The predicted amino acid sequence encodes a protein of approximately 26 kDa with homology to members of the NodL-LacA-CysE family of bacterial acetyltransferases. Southern blot analysis showed that genes similar to cap5H were present only in strains of S . aureus belonging to capsular serotypes 2, 4 and 5. In an in vitro assay, the parental strain was more resistant to opsonophagocytic killing than the mutant strain. In a mouse model of staphylococcal infection, the parental strain was able to seed the bloodstream from the peritoneal cavity and colonize the kidneys more efficiently than the O-deacetylated mutant. When cap5H was provided to the mutant in trans , it fully restored CP5 O-acetylation. The virulence of the complemented mutant strain closely approximated that of the parental strain.     

Phenotypic heterogeneity and temporal expression of the capsular polysaccharide in Staphylococcus aureus

Bacteria respond to ever‐changing environments through several adaptive strategies. This includes mechanisms leading to a high degree of phenotypic variability within a genetically homogeneous population. In Staphylococcus aureus, the capsular polysaccharide (CP) protects against phagocytosis, but also impedes adherence to endothelial cells and/or matrix proteins. We analysed the regulation of core biosynthesis genes (capA‐P) necessary for CP synthesis using single‐cell assays (immunofluorescence and promoter‐activity). In persistent human carriers, we found a distinct subpopulation of nasal S. aureus to be CP positive. In vitro, cap expression is also heterogeneous and strongly growth‐phase dependent. We asked whether this peculiar expression pattern (earlyOff/lateHeterogen) is orchestrated by the quorum system Agr. We show that the Agr‐driven effector molecule RNAIII promotes cap expression largely via inactivation of the repressor Rot. High NaCl, deletion of CodY or Sae also resulted in higher cap expression but did not change the earlyOFF/lateHeterogen expression pattern. Activity of the quorum system itself is largely homogenous and does not account for the observed heterogeneity of cap expression or the strictly growth phase dependent expression. Our findings are in contrast to the prevailing view that quorum sensing is the main driving force for virulence gene expression when bacterial cell densities increase.     

Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein–protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.     

Staphylococcus aureus growth and type 5 capsular polysaccharide production in synthetic media

The production of type 5 capsular polysaccharide by Staphylococcus aureus in synthetic media was investigated. The influence of medium components on capsular polysaccharide synthesis appeared to relate to the presence or absence of the component rather than to concentration gradient. The production of type 5 capsular polysaccharide was linked to energy availability and energy source, but not to carbohydrate concentration or carbon/nitrogen ratio. Regulation of capsular polysaccharide production by S. aureus in response to medium changes would appear to differ from that typically displayed in other organisms that produce polysaccharides.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Staphylococcus aureus growth and type 5 capsular polysaccharide production in synthetic media.

The production of type 5 capsular polysaccharide by Staphylococcus aureus in synthetic media was investigated. The influence of medium components on capsular polysaccharide synthesis appeared to relate to the presence or absence of the component rather than to concentration gradient. The production of type 5 capsular polysaccharide was linked to energy availability and energy source, but not to carbohydrate concentration or carbon/nitrogen ratio. Regulation of capsular polysaccharide production by S. aureus in response to medium changes would appear to differ from that typically displayed in other organisms that produce polysaccharides.     

Conjugation of capsular polysaccharide to alpha-haemolysin from Staphylococcus aureus as a glycoprotein antigen

The capsular polysaccharides of two pathogenic strains of Staphylococcus aureus 8914 and 31 were isolated and purified. These polysaccharides were conjugated to alpha-haemolysin prepared from the same strains. Amongst the various coupling procedures which were tested the best results were obtained with sodium cyanoborohydride and glutaraldehyde. The conjugates were purified and their immunologic properties were tested. They gave a positive response against antisera from whole bacterial cells.     

Production of type 5 capsular polysaccharide by Staphylococcus aureus grown in a semi-synthetic medium

The concentration of the type 5 capsular polysaccharide (CP) antigen of Staphylococcus aureus can be measured directly in cultures or cell suspensions by a two-step inhibition enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies. CP was synthesized during growth on a variety of carbon substrates and its production was not affected by the nature of the carbon source. High levels of yeast extract inhibited CP formation. CP was synthesized in batch culture at the same rate during exponential growth as in the post-exponential phase. Post-exponential CP production contributed at least half the final amount of CP measured. This phenomenon was observed in different culture media, although the specific yield of polysaccharide varied from one medium to another. Post-exponential CP production was observed in the pH range 6-7, but not at pH 8. Post-exponential production was strictly dependent on oxygen availability and did not occur under anaerobic conditions.     

Purine biosynthesis in Staphylococcus aureus

Summary The auxanographic analysis of 67 purine-dependent mutants and chromatographic analysis of their culture fluids were used to study purine biosynthesis in Staphylococcus aureus. The de novo biosynthesis of IMP from SAICAR, and the conversion of IMP to AMP and GMP were shown to occur via the conventional pathways reported for other organisms. Mutants blocked prior to the formation of SAICAR could not be differentiated by the tests used, and no substantial information on this portion of the pathway was obtained. The auxanographic characteristics of double mutants requiring both histidine and purines provided evidence that the sole route whereby S. aureus can convert AMP to IMP (and hence to GMP) is via those reactions of the histidine biosynthetic pathway leading to the formation of IGP and AICAR. In addition, we were able to mutationally separate AICAR transformylase and inosinocase; this separation has not been accomplished in other microorganisms.     

金黄色葡萄球菌基因调节系统研究进展

金黄色葡萄球菌是人类的一种重要病原菌,可以引起许多临床表现不同的感染性疾病。它所致感染的多样性和严重度取决于不同毒力因子的协同表达,而这些数量众多的毒力因子的表达会受到不同调节系统的控制,同时这些调节系统之间也存在着复杂的相互作用关系。这些基因调节系统主要有两大类：一类是双组分信号转导系统（如Agr、SaeRS、SrrAB、ArlSR、LytRS、WalKR）;另一类是转录因子f如Sar、Rot、MgrA、SigmaB）。它们的协同作用有助于金黄色葡萄球菌对外界环境信号做出反应,调节致病过程中毒力因子在不同情况下的表达。     

金葡菌荚膜多糖血清型的研究进展

金葡菌是引起奶牛乳腺炎的一种主要的致病菌,致病性金葡菌多数含有荚膜成分,金葡菌荚膜多糖有11种血清型。从不同血清型荚膜多糖的结构,基因构成,抗吞噬作用,致病性等方面作了介绍,并简要介绍了金葡菌荚膜多糖血清型的国内外研究现状。