| Summary: | In this work, two pathogenic microorganisms, Streptococcus pneumoniae and Leishmania parasites, are studied using AFM force spectroscopy with chemically modified tips in order to characterize their adhesive properties. In the work on S. pneumoniae, neutral hydrophobic and negatively-charged hydrophilic probes are used to detect non-specific forces on capsulated and unencapsulated type 2 bacteria. This work confirms the role of capsular polysaccharide in masking binding sites through a combination of steric hindrance and electrostatic repulsion, and shows that under the capsular surface there are pneumococcal molecules capable of hydrophobic binding. These results provide evidence that capsular reduction is important for achieving binding to the extracellular matrix of mucosal cells, and a good capsule coverage is essential to prevent binding of opsonins and detection by the immune system. It also supports the proposal that weak non-specific interactions act to reinforce and stabilize stronger, specific binding interactions when S. pneumoniae colonizes the nasopharynx. In the work on Leishmania parasites, specific sugar-binding interactions are investigated using galactose- and glucose-glycopolymer probes and Leishmania mexicana (two morphological forms of wild type parasites, an LPG- deficient mutant and its add-back), as well as one form of Leishmania major. The glycopolymer probes are developed and characterized using surface analysis techniques including FTIR-ATR, ellipsometry and optical tensiometry, and are shown to bind successfully and specifically to appropriate lectin-coated surfaces in an AFM assay. The experiments on the parasites show that sand fly midgut-adhesive forms of Leishmania mexicana interact strongly with both types of glycopolymer. They also show that this adhesion is lifecycle-stage specific, with infectious parasites exhibiting significantly reduced adhesion levels. Glycopolymer - parasite binding is also found to be LPG-dependent, with the LPG-deficient L. mexicana mutant and midgut-adhesive L. major (which has different, bulkier LPG) showing interaction levels comparable to the infectious lifecycle form. These results support the proposal that sugar moieties on the midgut epithelium of permissive sand flies are important for disease transmission. This work therefore confirms that AFM is a useful tool to quantitatively evaluate the interaction strength between selected biomolecules and different microorganisms, and can be tailored to a specific biological question: in this case providing valuable information about host colonization by S. pneumoniae and transmission of Leishmania parasites.
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