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Lecture 2

Notes Week 2

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MICR 4010
Rob Foster

Pathogenic Bacteriology – Week 2 Surfaces to which bacteria adhere The body provides 3 main types of surface: mucosa, skin, teeth. Mucosa exists in a variety of forms according to the body site. - Skin: keratinized cells, dry, shedding surface - Mucosa: moist, shedding, may be ciliate. It is a key barrier to infections, trapping microbes and bringing them back up. - Teeth: bacteria do not stick directly to enamel, a “conditioning film” of adsorbed molecules is always present on the surface (e.g. salivary glycoproteins). It is to the conditioning film that the first cells of the biofilm attach. - Internal body surfaces: when a bacterium is able to gain access to normally sterile sites, it may bind (I) directly to the tissue of a target organ, (II) to polymers of the extracellular matrix (ECM). Forces of attraction The average bacterial cell has 2um in diameter. Van der Waal forces - Natural attractive of repulsive forces (that are not ionic or electrostatic) between molecules. - They are a consequence of dipole-dipole/dispersion interactions, that set up synchronized attraction between closely-aligned molecules. Electrostatic forces - Arise from the forces that electric charges exert on one another. - In a hydrogen atom, the strength of the electrostatic interactions between proton and electron is 40x that of the gravitational force (very powerful). Hydrophobic interactions - Formed when non-polar molecules of the bacterium and substratum are in close proximity. - Intervening layers of water are dispersed - Adhesion becomes energetically favourable Cation bridging - In an aqueous environment: the surfaces of bacteria and most substrate carry a net negative charge. - Mutual repulsion ca be counteracted by divalent metal ions, e.g. Ca . 2+ - They act as bridge between the 2 negative surfaces. Specific interactions Receptor-ligand binding A molecule on the surface of the substratum (receptor) can recognize a molecule (ligand) on the bacterial surface with a complementary structure.  The bond formed is strong, and usually involves only a small portion of each of the molecules involved (epitopes).  Receptors and ligands can be proteins, polysaccharides, glycoproteins, glycolipids.  If interaction is between a protein and a carbohydrate epitope, this is known as lectin binding. Lectin Binding Importance: almost all microorganisms express surface-exposed carbohydrates; every surface- exposed carbohydrate is a potential lectin-reactive site; the specificity of lectin binding can be exploited (many MO can be identified according to their reactions with specific lectins). Bacterial structures involved in adherence Every known structure on the surface of a bacterium has the potential to act as an adhesion! Bacterial adhesins can be broken up into groups: frimbriae/pili, curli, flagella, S-layer, Capsule, cell wall, fibrils. FIMBRIAE/PILI Classified into 7 different types, distinguished by length, width and biochemical interactions. - Type I = very common in Enterobacteriaceae - Type IV = common to Enteropathogenic E. coli, Pseudomonas, Vibrio and Neisseria (gram positives and negatives). These are defined as specialized adherence organelles. Type I fimbriae Thinner, shorter and more numerous than flagella. Brittle (quebradiço) Can be found on the surfaces of both pathogenic and non-pathogenic species. Assembled by the chaperone-usher pathway of secretion Chaperone-usher pathway - It is GSP-dependent - When pilin subunits are secreted into the periplasm space but no chaperones are available, the protein aggregate and are degradated. - - If chaperone protein is available and secreted into the periplasmic space, then it binds to pilin subunit and stabilizes it, preventing it from aggregating with other subunits. - This type of binding is termed “donor strand complementation”. - - The chaperone-pilin subunits are then targeted to a pore in the OM called the “usher” (high affinity). - The usher facilitates removal of the chaperone’s donor strand from the pilin protein, replacing it with the NH do2ain of another pili protein  termed “donor strand exchange” - Once outside the cell, the enfolded pilin proteins form a helical structure – the pili. - Fim1 – an example of Type I fimbriae It is a key virulence determinant of pathogenic E. coli and Salmonella enterica. Main structural pilus subunit FimA -> polymerizes to form a thick, rigid rod. Subunits FimF & FimG, and adhesion FimH -> found a the distal end and form a flexible distal tip fibrillum (very flexible). During fimbriae biogenesis, the free N-terminal end of the last-placed subunit directs the assembly of the next-placed subunit. Type IV fimbriae Readily aggregate into bundles -> referred to as “bundle forming pili”. Allow the formation of “microcolonies” of expressing bacteria on surfaces. They are the only fimbriae that can be found in both gram positives and gram negatives. They are found in many different species, and are the most widespread organs of bacterial attachment. How they look like: - Extremely thin - Long - Flexible - Usually twine themselves together to form rope-like structures. Type IV fimbriae biogenesis - Pilin subunits always display a conserved N-terminal motif - They are synthesized as ‘prepilins’ - Targeted to the inner membrane and secreted through the Sec pathway. - An inner membrane peptidase then processes the protein and prepares it for filament assembly - Exact method of assembly poorly understood -> requires energy, couples chemical energy (ATP) to mechanical energy. - This assembly is reversible! Constant retraction and extrusion of pili filaments allows for a particular form of motility  twitching motility BFP – bundle forming pilus in EPEC Responsible for microcolony formation, occurring in a very short window in time. A possible further role for BFP -> appears to stabilize pockets of lipids on host cells where the bacteria adhere. If the host cells are depleted of lipid with chemicals, then the bacteria cannot adhere without the help of BFP. BFP help open up clearer communication channels between bacteria and host cell. Toxin co-regulated pilus of Vibrio cholerae Type IV pilus, essential to colonization of intestinal tract. No receptor has yet been found on host cells, but the pilin serves as a receptor for a bacteriophage (CTX), that carries the gene coding for cholera toxin (pilin acting as an adhesion for a bacteriophage). Another bacteriophage (VPI) carries the genes encoding the toxin co-regulated pilus, that are found on a large pathogenicity island incorporated to the bacteriophage genome. The toxin co- regulated pilus turns out to be the coat protein of this phage! CURLI Curli are the major protein subunit of a complex extracellular matrix produced by many Enterobacteriaceae (stick the bacteria together). Involved in adherence, invasion, aggregation, biofilm formation. Structurally and biochemically, they belong to a class of fibers known as amyloids  amyloid fiber formation is important in Alzheimer’s, Huntington’s and prion diseases. - CsgA is the major structural subunit (red squares) - CsgB is the nucleator protein Curli are part of the bacterial extracellular matrix Expression of curli and cellulose determines bacterial morphotype. Regulation of curli genes is complex and responds to environmental cues. FLAGELLA as an adhesin Flagella negative strains of several pathogens show a decreased level of adherence to host tissues. S-LAYER as an adhesin A slime layer is one form of bacterial glycocalyx. The fibers of the slime layer are loosely associated with the bacteria. The S-layer of Streptococcus mutans allows the pathogen to adhere to tooth enamel. Is also instrumental in the formation of oral biofilms (plaque). CAPSULE as an adhesin Capsule is another form is bacterial glycocalyx Glycoprotein fibers are more closely associated with the cell-wall than for S-layer. Example: hyaluronic acid capsule of Group A Streptococci  attaches to the host cell receptor CD44 Shielding of specific adhesion (proteção de adesão específica) The expression of bacterial glycocalyx is tightly regulated, it can also inhibit specific adhesion to certain surfaces -> masking of adhesins. This helps the bacterium to control its adhesion during phases of i
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