We describe the development, characterization, and biological testing of anew type of linear molecularly imprinted polymer (LMIP) designed to act as an anti-infective by blocking the quorum sensing (QS) mechanism and so abrogating the virulence of the pathogen Streptococcus pneumoniae.T he LMIP is prepared (polymerized) in presence of at emplate molecule,b ut unlike in traditional molecular imprinting approaches,n oc ross-linker is used. This results in soluble low-molecular-weight oligomers that can act as atherapeutic agent in vitro and in vivo.The LMIP was characterized by mass spectrometry to determine its monomer composition. Fragments identified were then aligned along the peptide template by computer modeling to predict the possible monomer sequence of the LMIP.T hese findings provide aproof of principle that LMIPs can be used to blockQS, thus setting the stage for the development of LMIPs an ovel drugdiscovery platform and class of materials to target Grampositive pathogens.
Super-resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. We developed µMagnify, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. We formulated an enzyme cocktail specifically designed for robust cell wall digestion and expansion of microbial cells without distortion while efficiently retaining biomolecules suitable for high-plex fluorescence imaging with nanoscale precision. Additionally, we developed an associated virtual reality tool to facilitate the visualization and navigation of complex three-dimensional images generated by this method in an immersive environment allowing collaborative exploration among researchers around the world. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables development of new diagnosis strategies against infectious diseases.
Extracellular vesicles (EVs) represent a highly sophisticated cell-to-cell mailing system across all biological kingdoms. EVs have long been characterized from many Gram-negative species and, recently from Gram-positive bacteria, including the major respiratory pathogen Streptococcus pneumoniae (pneumococcus). Our studies reveal that pneumococcal-derived vesicles can be internalized by macrophages, T cells, and epithelial cells. In vitro, EVs induce cytokine signaling in macrophages, including dose-dependent NF-kB signaling in murine RAW 264.7 and human primary macrophages. When administered systemically into a mouse, pneumococcal EVs result in splenomegaly and induced a sepsis-like cytokine storm. When immobilized in a hydrogel implant for local administration into a mouse, pneumococcal EVs recruited lymphocytes and macrophages. Moreover, pneumococcal lipoproteins are major contributors to NF-kB signaling and inflammatory responses, as these phenotypes were substantially reduced with EVs from a lipoprotein deficient strain (Δlgt) as compared to EVs from the wildtype strain. Taken together, in vivo studies suggest that pneumococcal vesicles alone are sufficient to induce inflammatory responses and tissue damage in mammalian hosts. Overall, our data suggest that pneumococcal EVs display potent immunomodulatory effects on host immune cells highlighting their pivotal role during the infectious process, either by manipulating host responses or by triggering host-defense systems. Thus, pneumococcal EVs are virulence determinants and may be effective tools for vaccine development.
Extracellular vesicles (EVs) are complex, cell‐derived nanoparticles generated by all cell types. EVs are composed of lipid bilayer membranes and their associated membrane proteins, nucleic acids, and luminal proteins. The mechanism by which Gram‐positive bacteria shed EVs is still unknown. EVs from the Gram‐positive human pathogen S. pneumoniae, which is a major cause of otitis medi and pneumonia, are of particular interest because of how they EVs modulate the host immune response. To uncover possible mechanisms for EV production and shedding in S. pneumoniae, we have performed a comparative proteomics analysis of EV membrane proteins versus whole‐cell membrane proteins. Membrane proteins were enriched from intact S. pneumoniae cells or their EVs using a ProMTag labeling and capture workflow. ProMTag is a bifunctional protein tag where one moiety of the tag is able to form a reversible, covalent link to primary amines on proteins. The other moiety is methyltetrazine, which can form an irreversible, covalent bond with trans‐Cyclooctene (TCO) on the surface of beads to capture ProMTagged proteins for cleanup and elution. Using this workflow plasma membrane proteins can be tagged, captured, washed to remove non‐plasma membrane proteins, and then eluted in their original, unmodified state. In this study, intact cells and EVs from S. pneumoniae cultures were separated and the extracellular domains of membrane proteins in these two fractions were labeled with ProMTag. The membrane proteins were then enriched, washed, and eluted using the ProMTag workflow. These membrane protein populations were then TMT labeled and analyzed using mass spectrometry. Comparative analysis revealed membrane proteins that are concentrated or absent in EV membranes relative to bulk plasma membrane from whole cells, indicating a selective process for EV formation in S. pneumoniae. With this information, we present a new model for EV formation and shedding in S. pneumoniae.
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