MICR 221 - Lecture 13: Adherence and Colonization PDF

Lecture 13: Adherence and Colonization Feb. 4, 2025 1 Lecture Learning Outcomes After this lecture, students will be able to describe… The structures of bacterial capsules, and how they are made How capsules protect bacteria from stresses, including innat...

Lecture 13: Adherence and Colonization Feb. 4, 2025 1 Lecture Learning Outcomes After this lecture, students will be able to describe… The structures of bacterial capsules, and how they are made How capsules protect bacteria from stresses, including innate and adaptive immunity The structure and function of chaperone-usher pili, and how they are assembled How pili contribute to bacterial virulence, and how they are targeted by the immune system How pili on Gram-positive bacteria are made 2  Bacterial Adherence Ability to adhere is helpful in many environments Adherence to food sources E.g., cellulolytic microbes and cellulose Adherence to host cells, tissues Commensals, pathogens Adherence to abiotic surfaces E.g., biofilms on medical devices 3 Image from: https://biofilm.montana.edu/multimedia/images/index.html  Glycocalyx Bacteria often have a glycocalyx Polysaccharide layer capsule surrounding cell Contributes to adherence Related to extracellular polymeric substances (EPS) in biofilms Slime layer Diffuse surface layer Easily removed from cell Capsule Organized surface layer Firmly attached to cell 4 Image from: https://doi.org/10.1128/AEM.02075-07  Capsules Surface layer of polysaccharides Usually… Long polymers covalently attached to cell surface Can be attached to LPS, phospholipids, peptidoglycan Not required for growth in the lab Often observed for bacteria in natural environments Protection Visualized by negative staining Many stains don’t bind to capsules 5 Image from: https://doi.org/10.1021/acs.langmuir.8b04122  Capsular Polysaccharides Each strand is 1 - 100 kDa Multiple repeating sugar subunits Similar to LPS O-antigen Structure varies between strains, species Different sugars, modifications, branching patterns >80 E. coli capsular serotypes E.g., E. coli K5 capsular polysaccharide ~250 repeats of disaccharide subunit Attached to phosphatidylglycerol (in outer membrane) phosphatidylglycerol 6 Image from: https://doi.org/10.3390/biom10111516  Capsule Functions Adherence Protects against desiccation Highly hydrated Can be used as nutrient source Prevents engulfment by predators (e.g., protozoa) Protects against bacteriophage infection Reduces efficacy of antibiotics, antimicrobial peptides 7 Image from: https://fineartamerica.com/featured/skin-bacterium-enclosed-in-a-capsule-dr-kari-lounatmaa.html  Capsules and Virulence Virulence factor (recall: Griffith mouse experiments) Protects against innate immune system, preventing: Complement activation, inflammation Formation of membrane attack complex (MAC) Opsonization, phagocytosis Major contributor to serum resistance Ability to survive and spread in blood Leads to bacteremia, meningitis, etc 8 Image from: https://doi.org/10.1098/rstb.2016.0221  Capsules and the Immune System Adaptive immune response often depends on antibodies Opsonization However, capsules protect bacteria from antibodies Mask other antigenic components Some capsules made of sugars that are common in the host (e.g., hyaluronic acid, sialic acid) Not immunogenic 9 Image from: https://open.oregonstate.education/microbiology/chapter/18-1overview-of-specific-adaptive-immunity/  Capsular Vaccines Capsules are major vaccine targets Accessible to immune system Capsular vaccines are very effective * * * But, polysaccharides alone not very immunogenic Must be modified to evoke immune response Conjugate vaccines: capsular polysaccharides attached to immunogenic protein carrier 10 Image from: https://www.kflaph.ca/en/resource-catalogue/Pneumococcal-Conjugate-Vaccine.aspx Capsule Assembly Capsule components must be transported to surface Sugars activated with nucleoside diphosphate group UDP-sugar pyrophosphorylase makes UDP-sugar Sugar then transferred to undecaprenyl phosphate 11 Roles of Undecaprenol Helps hydrophilic sugars travel through hydrophobic membrane bilayer Also used to make peptidoglycan, LPS, teichoic acids Keeps sugars close to membrane (and to enzymes) Activates sugars (reactive pyrophosphate group) 12 Capsule Assembly in Gram-Positives Subunit assembled in cytoplasm Flipped to outer face by flippase Polymerized by polymerase Attached to surface molecule (e.g., peptidoglycan) 13  Capsule Assembly in Gram-Negatives Some use Wzx-Wzy-dependent pathway Subunit assembled in cytoplasm (glycosyltransferases, GTs) Flipped across membrane by Wzx (flippase) Polymerized by Wzy (polymerase) Translocated to surface through Wza channel Incorporated on cell surface 14 Image from: DOI 10.1146/annurev.biochem.75.103004.142545  Capsule Assembly in Gram-Negatives Some Gram-negatives don’t use undecaprenol Can use ABC transporter-dependent pathway Assemble full polysaccharide in cytoplasm with GTs Transfer to lipid (e.g., diacylglycerol phosphate) Polysaccharide and lipid transported to surface by ABC transporter (e.g., KpsM, KpsT) 15 Image from: DOI 10.1146/annurev.biochem.75.103004.142545  Bacillus anthracis Capsule Some capsules not made from sugars Bacillus anthracis has a proteinaceous capsule Long D-glutamic acid (amino acid) polymers Important virulence factor Major role in anthrax Capsule helps B. anthracis evade host immunity Interferes with complement activation Prevents phagocytosis Non-immunogenic 16 Image from: https://doi.org/10.3390/microorganisms8121864 Non-Flagellar Appendages Bacteria covered in rigid hairlike structures Thin (3 μm), thin (~1 nm) Flexible Adhesins attached to ends Similar roles as Gram- negatives Adhesion, biofilm formation Unlike Gram-negatives: Subunits covalently attached together Pili are attached to peptidoglycan 30 Assembly of Gram-Positive Pili Subunits transported by general secretion (Sec) system Pilus subunits contain cell wall sorting signal Positively charged (stays near membrane) LPXTG motif Sortase cuts LPXTG, forms complex with pilus subunit 31 Gram-Positive Pilus Assembly Pilus-specific sortases form polymers of pilus subunits Covalent bonds confer mechanical strength Housekeeping sortase then transfers pilus to lipid II Lipid II (+ pilus) incorporated into PG by PBPs 32 Reminders Bacteriology Quiz 3 closes Feb. 4 at 3 PM Bacteriology Quiz 4 opens Feb. 6 at 3 PM Lectures 12 - 14 Office Hour Tuesday, 12 – 1 PM, Botterell Room 449 Lab 2 Assignment Section 002: due Feb. 6, 2:30 PM Lab 3 Section 002: Feb. 6 at 2:30 PM Section 004: Feb. 6 at 4:00 PM Sections 003, 005: Feb. 7 at 2:30 PM Complete Lab 3 Pre-Lab Quiz before 33