Mjsabulski

Motile bacteria navigate chemical environments by using chemoreceptors. The output of these protein sensors is linked to motility machinery and enables bacteria to follow chemical gradients. Understanding the chemical specificity of different families of chemoreceptors is essential for predicting and controlling bacterial behavior in ecological niches, including symbiotic and pathogenic interactions with plants and mammals. The identification of chemical(s) recognized by specific families of receptors is limited by the low throughput and complexity of chemotaxis assays. To address this challenge, we developed a microfluidic-based chemotaxis assay that is quantitative, simple, and enables high-throughput measurements of bacterial response to different chemicals. Using the model bacterium Escherichia coli, we demonstrated a strategy for identifying molecules that activate chemoreceptors from a diverse compound library and for determining how global behavioral strategies are tuned to chemical environments.

Marvels of microfluidics: We report a simple microfluidic-based high-throughput assay to screen large chemical libraries for bacterial chemotaxis activity. We provide experiments that demonstrate how our approach can be used to rapidly identify and characterize the chemotactic behavior of uncharacterized bacterial species.

All Enterobacteriaceae express a polysaccharide known as enterobacterial common antigen (ECA), which is an attractive target for the development of universally acting immunotherapies. The first chemical synthesis of ECA-derived oligosaccharides for the development of such therapies is described. A number of synthetic challenges had to be addressed, including the development of concise synthetic procedures for unusual monosaccharides, the selection of appropriate orthogonal protecting groups, the development of stereoselective glycosylation methods, appropriate timing for the introduction of the carboxylic acid groups on the ManpNAcA moieties, and the selection of appropriate conditions for the reduction of multiple azido moieties. The synthetic compounds were employed to uncover immunodominant moieties of ECA. Furthermore, a monoclonal antibody (mAb) was developed that binds to ECA and can selectively recognize a wide range of Enterobacteriaceae species.

Hitting the sweet spot: All Enterobacteriaceae express the polysaccharide enterobacterial common antigen (ECA), which is an attractive target for the development of universally acting immunotherapies. ECA-derived oligosaccharides were chemically synthesized and used to uncover immunodominant epitopes and develop a monoclonal antibody showing broad recognition of Enterobacteriaceae.

Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6-, 3,4-, or 2,3-positions. A trans-2,3-linked pyruvate is present on the CPS of the Gram-positive bacterium Streptococcus pneumoniae serotype 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4 CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.

Unusually Protective. In the search for a glycan epitope for protection against Streptococcus pneumoniae serotype 4, total synthesis of the repeating unit (see structure) enabled the identification of pyruvate as a crucial determinant of immunogenicity. This lays the foundation for the development of a new antibacterial vaccine candidate against Streptococcus pneumoniae serotype 4.

Here the synthesis and characterization of a new class of spiropyran-based protease inhibitor is reported that can be reversibly photoswitched between an active spiropyran (SP) isomer and a less active merocyanine (MC) isomer upon irradiation with UV and visible light, respectively, both in solution and on a surface of a microstructured optical fiber (MOF). The most potent inhibitor in the series (SP-3 b) has a C-terminal phenylalanyl-based α-ketoester group and inhibits α-chymotrypsin with a K_i of 115 nM. An analogue containing a C-terminal Weinreb amide (SP-2 d) demonstrated excellent stability and photoswitching in solution and was attached to the surface of a MOF. The SP isomer of Weinreb amide 2 d is a competitive reversible inhibitor in solution and also on fiber, while the corresponding MC isomer was significantly less active in both media. The ability of this new class of spiropyran-based protease inhibitor to modulate enzyme activity on a MOF paves the way for sensing applications.

Switch the enzyme activity on and off: The first examples of spiropyran-based protease inhibitors are reported. Here the inhibitory activity can be controlled by irradiation with light of a specific wavelength, both in solution and on attachment to an optical fiber. The unique ability to modulate enzyme activity in nanoliters of sample fluids with a fiber provides an important basis for biosensing applications.

Lasiocepsin is a unique 27-residue antimicrobial peptide, isolated from Lasioglossum laticeps (wild bee) venom, with substantial antibacterial and antifungal activity. It adopts a welldefined structure consisting of two α-helices linked by a structured loop. Its basic residues form two distinct positively charged regions on the surface whereas aliphatic side chains contribute to solvent-accessible hydrophobic areas, thus emphasising the amphipathic character of the molecule. Lasiocepsin structurally belongs to the ShK family and shows a strong preference for anionic phospholipids; this is further augmented by increasing concentrations of cardiolipin, such as those found at the poles of bacterial cells. The membrane-permeabilising activity of the peptide is not limited to outer membranes of Gram-negative bacteria. The peptide interacts with phospholipids initially through its N terminus, and its degree of penetration is strongly dependent on the presence of cardiolipin.

Membrane penetration: The amphipathic character of lasiocepsin is key for its integration into bacterial membranes. The peptide shows a strong preference for anionic phospholipids, and this is further stimulated by increasing concentrations of cardiolipin at the poles of bacterial cells. Cardiolipin-containing model membranes can be fully penetrated by the peptide.