Bacterial Capsules, Pili, and Adhesins

• Lecture topic is adherence and colonization
• Lecture date is February 4, 2025

Lecture Learning Outcomes

• Structures of bacterial capsules can be described
• How bacterial capsules are made can be described
• How capsules protect bacteria from stresses, including innate and adaptive immunity can be described
• Structure and function of chaperone-usher pili can be described
• How chaperone-usher pili are assembled can be described
• How pili contribute to bacterial virulence can be described
• How pili are targeted by the immune system can be described
• How pili on Gram-positive bacteria are made can be described

Bacterial Adherence

• The ability to adhere is helpful in many environments
• Bacteria can adhere to food sources
• Cellulolytic microbes and cellulose are examples of sources bacteria will adhere to
• Bacteria can adhere to host cells and tissues
• Commensals and pathogens are tissues that bacteria adhere to
• Bacteria can adhere to abiotic surfaces
• Biofilms on medical devices are an instance of abiotic surfaces bacteria adhere to

Glycocalyx

• Bacteria often possess a glycocalyx
• This is a Polysaccharide layer surrounding cell
• It contributes to adherence
• It is related to extracellular polymeric substances (EPS) in biofilms
• Slime layer is a diffuse surface layer
• It's easily removed from cell
• Capsules are Organized surface layer
• They are firmly attached to cell

Capsules

• Capsules are surface layers of polysaccharides
• They are usually long polymers covalently attached to cell surface
• Capsules can be attached to LPS, phospholipids, or peptidoglycan
• Capsules aren't required for growth in the lab
• Capsules are often observed in bacteria in natural environments
• Capsules provide Protection
• They are visualized by negative staining
• Many stains don't bind to capsules

Capsular Polysaccharides

• Each strand is 1 - 100 kDa
• Multiple repeating sugar subunits present
• It is similar to LPS O-antigen
• Structure varies between strains, species
• Have different sugars, modifications, branching patterns
• There are >80 E. coli capsular serotypes
• E.g., E. coli K5 capsular polysaccharide
• Has ~250 repeats of disaccharide subunit
• The capsule is attached to phosphatidylglycerol (in outer membrane)

Capsule Functions

• Capsules provide adherence
• The capsule protects against desiccation
• The capsule is highly hydrated
• The capsule can be used as nutrient source
• It prevents engulfment by predators (e.g., protozoa)
• It protects against bacteriophage infection
• It reduces efficacy of antibiotics, antimicrobial peptides

Capsules and Virulence

• Capsules are virulence factors
• Protects against innate immune system, preventing:
• Complement activation and inflammation
• Formation of membrane attack complex (MAC)
• Opsonization and phagocytosis
• They are Major contributors to serum resistance
• Capsules provide the Ability to survive and spread in blood
• Capsules leads to bacteremia, meningitis, etc

Capsules and the Immune System

• Adaptive immune response often depends on antibodies
• Opsonization
• Capsules protect bacteria from antibodies
• They mask other antigenic components
• Some capsules made of sugars that are common in the host (e.g., hyaluronic acid, sialic acid)
• These capsules are not immunogenic

Capsular Vaccines

• Capsules are major vaccine targets
• Capsules are accessible to immune system
• Capsular vaccines are very effective
• Polysaccharides alone not very immunogenic
• Must be modified to evoke immune response
• Conjugate vaccines: capsular polysaccharides attached to immunogenic protein carrier

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

Roles of Undecaprenol

• Helps hydrophilic sugars travel through hydrophobic membrane bilayer
• Used to make peptidoglycan, LPS, teichoic acids
• Keeps sugars close to membrane (and to enzymes)
• Activates sugars (reactive pyrophosphate group)

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)

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

Capsule Assembly in Gram-Negatives

• Some Gram-negatives don't use undecaprenol
• They 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)

Bacillus anthracis Capsule

• Some capsules not made from sugars
• Bacillus anthracis has a proteinaceous capsule
• Uses long D-glutamic acid (amino acid) polymers
• This is an important virulence factor
• This plays Major role in anthrax
• Capsule helps B. anthracis evade host immunity
• it Interferes with complement activation
• It Prevents phagocytosis
• It's Non-immunogenic

Non-Flagellar Appendages

• Bacteria covered in rigid hairlike structures
• These are Thin (<10 nm diameter), long (up to several µm)
• Many are present per cell
• Usually called pili (or fimbriae)
• They are Involved in adherence
• Adhesins
• Common in Gram-negatives
• Other adhesins more common in Gram-positives

Non-Flagellar Appendages

• Gram-negatives have different kinds of "pili”
• There are Type IV secretion pili (sex pili) for conjugation
• There are Type IV pili for twitching motility
• There are Type III secretion systems
• There are Chaperone-usher (CU) pili (most common)

Chaperone-Usher (CU) Pili

• Contain a Rigid helical structure
• It is made up of 1000s of major pilins in the Rod
• Ends in adhesin
• Adhesin and rod linked by minor pilins in the Fibrillum
• Name is based on how pilins are incorporated:
• Chaperones help pilins fold in periplasm
• Usher adds pilins to base of pilus

CU Pili and Adhesins

• Some CU pili are virulence factors
• They are Used to attach to host, form biofilms
• Attachment usually needed for colonization, infection
• Attachment Prevents bacteria from being washed away
• It's present E.g., in the GI, respiratory, and urinary tracts

Pili and Pathogenesis

• Adhesins only bind to specific molecules
• Often adheres to carbohydrates
• Determines which cells and tissues bacteria can attach to
• Influence where bacteria cause infections
• Streptococcus pyogenes (strep throat): pharyngeal epithelial cells
• Streptococcus pneumoniae: lung epithelial cells
• Uropathogenic E. coli (UPEC): uroepithelial cells

Uropathogenic E. coli (UPEC)

• UPEC causes ~90% of urinary tract infections (UTIs)
• Type I pili (T1P) attach to proteins on uroepithelial cells
• It is a type of CU pilus
• Causes cytoskeletal rearrangements, internalization
• Forms intracellular bacterial communities (IBCs)

UPEC and Pyelonephritis

• UPEC can ascend ureters, enter kidneys
• Can cause upper UTIs, pyelonephritis (inflammation of kidney)
• Use P pili to attach to glycolipids on kidney epithelial cells
• This uses a CU pilus
• To reach and colonize upper urinary tract:
• Downregulate T1P production
• Upregulate P pilus production

Pili and the Immune System

• Targeted by immune cells
• Surface-exposed
• Many copies of pilin proteins
• Purified adhesins are potential vaccines
• Generates antibodies that bind to adhesins
• Prevent attachment, colonization

Antigenic Variation

• Capsule can change pilus structure to evade immune system
• Antigenic variation
• Neisseria gonorrhoeae:
• Pilin gene pilE is transcribed
• Other pilin genes not transcribed (pilS)
• Recombination of pilE and pilS creates new pilE variants

P Pilus Assembly

• P pilus subunits exported to periplasm by general secretion (Sec) system
• Transports unfolded proteins
• PapD binds to pilus subunits, helps them fold
• Chaperone part of CU pilus

P Pilus Assembly

• PapC forms base of pilus
• Translocates subunits to surface
• Usher part of CU pilus
• PapD delivers subunits to PapC
• Fibrillum is assembled first:

P Pilus Assembly

• Next, Papa (major pilin) proteins translocated by PapC
• Added to base of growing pilus (cf. flagella)
• Mutants lacking PapA don't make pili, but are adherent

Regulation of Pilus Length

• PapH:
• Keeps pilus attached to cell
• Blocks PapA addition
• Mutants lacking PapH make long pili that are not attached to cell
• Pilus length determined by levels of PapA, PapH
• PapH overproduction results in shorter pili

Gram-Positive Pili

• Some Gram-positives have pili
• Long (>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

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

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

Explore the function of bacterial capsules and pili, including their role in adherence and protection against environmental stresses. Learn about the composition of capsules, the different types of pili, and the mechanisms of adhesion in bacteria.