A semisynthetic carbohydrate-lipid vaccine that protects against S. pneumoniae in mice.

Severe forms of pneumococcal meningitis, bacteraemia and pneumonia result in more than 1 million deaths each year despite the widespread introduction of carbohydrate-protein conjugate vaccines against Streptococcus pneumoniae. Here we describe a new and highly efficient antipneumococcal vaccine design based on synthetic conjugation of S. pneumoniae capsule polysaccharides to the potent lipid antigen α-galactosylceramide, which stimulates invariant natural killer T (iNKT) cells when presented by the nonpolymorphic antigen-presenting molecule CD1d. Mice injected with the new lipid-carbohydrate conjugate vaccine produced high-affinity IgG antibodies specific for pneumococcal polysaccharides. Vaccination stimulated germinal center formation; accumulation of iNKT cells with a T follicular helper cell phenotype; and increased frequency of carbohydrate-specific, long-lived memory B cells and plasmablasts. This new lipid-carbohydrate vaccination strategy induced potent antipolysaccharide immunity that protected against pneumococcal disease in mice and may also prove effective for the design of carbohydrate-based vaccines against other major bacterial pathogens.

The position of hydrophobic residues tunes peptide self-assembly.

The final structure and properties of synthetic peptides mainly depend on their sequence composition and experimental conditions. This work demonstrates that a variation in the positions of hydrophobic residues within a peptide sequence can tune the self-assembly. Techniques employed are atomic force microscopy, transmission electron microscopy and an innovative method based on surface acoustic waves. In addition, a systematic investigation on pH dependence was carried out by utilizing constant experimental parameters.

Measurement of porcine haptoglobin in meat juice using surface acoustic wave biosensor technology.

Aim of the study was the application of biosensor technique to measure the concentration of an acute phase protein (APP) within complex matrices from animal origin. For the first time, acute phase protein haptoglobin (Hp) was detected from unpurified meat juice of slaughter pigs by a label-free biosensor-system, the SAW-based sam®5 system. The system uses a sensor chip with specific antibodies to catch Hp while the mass-related phase shift is measured. The concentration is calculated as a function of these measured phase shifts. The results correlate very well with reference measurement results obtained by enzyme-linked immunosorbent assay (ELISA), R=0.98. The robust setup of the surface acoustic wave (SAW)-based system and its ability to measure within very short time periods qualifies it for large-scale analyses and is apt to identify rapidly pigs in the meat production process whose consumption would have an increased risk for consumers.

The E. coli effector protein NleF is a caspase inhibitor.

Enterohemorrhagic and enteropathogenic E. coli (EHEC and EPEC) can cause severe and potentially life-threatening infections. Their pathogenicity is mediated by at least 40 effector proteins which they inject into their host cells by a type-III secretion system leading to the subversion of several cellular pathways. However, the molecular function of several effectors remains unknown, even though they contribute to virulence. Here we show that one of them, NleF, binds to caspase-4, -8, and -9 in yeast two-hybrid, LUMIER, and direct interaction assays. NleF inhibits the catalytic activity of the caspases in vitro and in cell lysate and prevents apoptosis in HeLa and Caco-2 cells. We have solved the crystal structure of the caspase-9/NleF complex which shows that NleF uses a novel mode of caspase inhibition, involving the insertion of the carboxy-terminus of NleF into the active site of the protease. In conformance with our structural model, mutagenized NleF with truncated or elongated carboxy-termini revealed a complete loss in caspase binding and apoptosis inhibition. Evasion of apoptosis helps pathogenic E. coli and other pathogens to take over the host cell by counteracting the cell's ability to self-destruct upon infection. Recently, two other effector proteins, namely NleD and NleH, were shown to interfere with apoptosis. Even though NleF is not the only effector protein capable of apoptosis inhibition, direct inhibition of caspases by bacterial effectors has not been reported to date. Also unique so far is its mode of inhibition that resembles the one obtained for synthetic peptide-type inhibitors and as such deviates substantially from previously reported caspase-9 inhibitors such as the BIR3 domain of XIAP.

Kinetic binding analysis of aptamers targeting HIV-1 proteins by a combination of a microbalance array and mass spectrometry (MAMS).

An enhanced chip-based detection platform was developed by integrating a surface acoustic wave biosensor of the Love-wave type with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). The system was applied to characterize the interaction of aptamers with their cognate HIV-1 proteins. The aptamers, which target two proteins of HIV-1, were identified using an automated in vitro selection platform. For aptamers S66A-C6 and S68B-C5, which target the V3 loop of the HIV-1 envelope protein gp120, KD values of 406 and 791 nM, respectively, were measured. Aptamer S69A-C15 was shown to bind HIV-1 reverse transcriptase (HIV-1 RT) with a KD value of 637 nM when immobilized on the biosensor surface. HIV-1 RT was identified with high significance using MALDI-ToF MS even in crude protein mixtures. The V3-loop of gp120 could be directly identified when using chip-bound purified protein samples. From crude protein mixtures, it could be identified indirectly with high significance via its fusion-partner glutathione-S-transferase (GST). Our data show that the combination of the selectivity of aptamers with a sensitive detection by MS enables the reliable and quantitative analysis of kinetic binding events of protein solutions in real time.

Aptamers and biosensors.

The immobilization procedure to a biosensor surface has a major influence on the measurement results. To characterize the immobilization onto various biolayers, the interaction of DNA anti-thrombin aptamer with the protein thrombin was used as a model system. The aptamer was immobilized to a two-dimensional alkanethiol SAM via carboxylamide bonds and to a three-dimensional dextran matrix via streptavidin-biotin interaction. The calculated K (D) values of about 260 and 267 nM, respectively, were comparable, while the amount of bound analyte varied by a factor of 2, depending on the accessibility of the immobilized aptamer. Differences in the specificity were shown by use of the similar protein elastase.

Selection process generating peptide aptamers and analysis of their binding to the TiO2 surface of a surface acoustic wave sensor.

The set-up presented in this article is intended for the selection of peptides which serve as specific binders to suitable materials. Additionally, the interaction of such binders with material surfaces can be characterized. Using this approach, a subset of peptides which adhere to the mineral TiO(2) was generated by means of a cell surface display library. The peptides are constrained by a thioredoxin scaffold. Selection of proteins was carried out on a silicium wafer sputtered with TiO(2) in anatase conformation. To verify binders and to analyze the binding kinetics of the diluted suspension of the purified proteins, the chip-based S-sens K5 surface acoustic wave sensor system was used. The surface of the sensor chips was also TiO(2), resembling the material of the Si wafer selection target retaining the peptides. Several peptides were identified. The respective binding behavior differed. The data derived from real-time interaction analysis were evaluated to select for strong and specific binders. For one of these peptides, the binding kinetics was analyzed. On- and off-rate binding constants were extracted from the fitted curves. With the resulting association rate constant k(on) and the dissociation constant k(off), the affinity of the peptide for the TiO(2) surface was calculated, represented by the equilibrium dissociation constant K(D)=81 nM.

Surface acoustic wave biosensor as a tool to study the interaction of antimicrobial peptides with phospholipid and lipopolysaccharide model membranes.

Surface acoustic wave biosensors are a powerful tool for the study of biomolecular interactions. The modulation of a surface-confined acoustic wave is utilized here for the analysis of surface binding. Phase and amplitude of the wave correspond roughly to mass loading and viscoelastic properties of the surface, respectively. We established a procedure to reconstitute phospholipid and lipopolysaccharide bilayers on the surface of a modified gold sensor chip to study the mode of action of membrane-active peptides. The procedure included the formation of a self-assembled monolayer of 11-mercaptoundecanol, covalent coupling of carboxymethyl-dextran, and subsequent coating with a poly- l-lysine layer. The lipid coverage of the surface is highly reproducible and homogeneous as demonstrated in atomic force micrographs. Ethanol/triton treatment removed the lipids completely, which provided the basis for continuous sequences of independent experiments. The setup was applied to investigate the binding of human cathelicidin-derived peptide LL32, as an example for antimicrobial peptides, to immobilized phosphatidylserine membranes. The peptide-membrane interaction results in a positive phase shift and an increase in amplitude, indicating a mass increase along with a loss in viscosity. This suggests that the bilayer becomes more rigid upon interaction with LL32.

Surface acoustic wave sensors in the bioanalytical field: recent trends and challenges.

This is a comparison of the latest developments in the emerging field of surface acoustic wave (SAW) sensors. Progress has been made particularly with regard to (sub-) microstructure technology and material sciences. Improvements are displayed based on the impact on a new generation of SAW sensors working efficiently in liquid media, from modeling to the fabrication steps of the individual components. It is explained, which obstacles have to be overcome for applications to the bioanalytical field. SAW sensors are shown to be extremely useful for the analysis of both small and large molecules as well as whole cells interacting with an immobilized binding partner. The output signal gives information about the pure mass loading, intrinsic properties of bound materials, or viscoelastic effects like structural rearrangements. Different setups are shown that minimize the influence of physical bulk effects on the sensor signal, e.g. salt content and viscosity. The choice of materials which can be used for sensible surfaces are presented, enabling the development of completely new coupling chemistries. Finally, the advantages compared to other biosensor technologies are pointed out.

Mutations of the act promoter in Myxococcus xanthus.

Mutations within the -12 and -24 elements provide evidence that the act promoter is recognized by sigma-54 RNA polymerase. Deletion of the -20 base pair, which lies between the two conserved elements of sigma-54 promoters, decreased expression by 90%. In addition, mutation of a potential enhancer sequence, around -120, led to an 80% reduction in act gene expression. actB, the second gene in the act operon, encodes a sigma-54 activator protein that is proposed to be an enhancer-binding protein for the act operon. All act genes, actA to actE, are expressed together and constitute an operon, because an in-frame deletion of actB decreased expression of actA and actE to the same extent. After an initially slow phase of act operon expression, which depends on FruA, there is a rapid phase. The rapid phase is shown to be due to the activation of the operon expression by ActB, which completes a positive feedback loop. That loop appears to be nested within a larger positive loop in which ActB is activated by the C signal via ActA, and the act operon activates transcription of the csgA gene. We propose that, as cells engage in more C signaling, positive feedback raises the number of C-signal molecules per cell and drives the process of fruiting body development forward.

Discrimination of single mutations in cancer-related gene fragments with a surface acoustic wave sensor.

Here, we report on using a surface acoustic wave sensor for the highly sensitive and accurate detection of individual point mutations in cancer-related gene DNA fragments from single injections. Our sensor measures both the mass and viscosity signals and, thus, allows discriminating between mass effects resulting from hybridization of short DNA strands and viscosity effects due to increasing amounts of DNA deposited on the sensor. Single nucleotide exchanges or deletions are distinguished reliably and with exceeding simplicity from the wild-type sequences, on the basis of differences in their dissociation or association rates starting at low nanomolar concentrations. Mutant oligonucleotides were identified immediately from viewing the recorded signal and without further processing of the data. Multiple repeated binding cycles were possible over days without affecting sensitivity. To achieve signal amplification, our new bioassay can also apply multiple hybridization steps based on sandwich hybridizations. Kinetic evaluations gave insight into the physicochemical properties of the fragments used that explain the differences in their binding processes.

act operon control of developmental gene expression in Myxococcus xanthus.

Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the csgA gene, which encodes the C-signal, is directed by four genes of the act operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the act genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in actA, actB, actC, or actD mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null act mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The actA and actB null mutations substantially decreased the expression of all these reporters. The other act null mutations caused either premature expression to wild-type levels (actC) or delayed expression (actD), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.