Biofilms and Complement Resistance Flashcards
name 3 types of biofilms
- Mixed communities of interdependent bacteria (synotrophism)
- Single species biofilms
- Matrix of exopolysaccharide and/or DNA
whats the Structure of Biofilms
- Three-dimensional structure (flat or larger structures)
- Interspersed by water channels permitting circulation of nutrients and oxygen
- But variable environments (e.g. low oxygen in centre)
what is Exopolysaccharide
Secreted polysaccharides Wide array of types Some species produce only one type and others multiple types Often appear as mucoid colonies on plates, frequently slow-growing
give 2 examples of Exopolysaccharides
- staphylococci produce a slime substance PIA or PNAG
- E. coli produces colanic acid
how do Biofilms work in Pathogenesis
- Attachment to host tissues
- Increase metabolic efficiency of community Obtain a high density of microorganisms
- Gene transfer
- Protection from antimicrobial agents (antibiotics), immune effectors and phagocytosis
how do Biofilms provide Resistance to Antimicrobial agents
- Prevent antimicrobial agents from reaching bacterial cells
- Nutrient-starved, slow-growing bacterial cells are resistant to many antimicrobial agents
- Distinct biofilm-associated phenotypic state resistant to agents
- Source of re-infection after killing of planktonic cells
whats the prevalence of Cystic Fibrosis?
affects 70,000 people/annum worldwide
how does infection occur in cystic fibrosis
- Mutation in cystic fibrosis transmembrane conductance regulator (CFTR)
- CFTR-defect lung leads to defects in mucociliary clearance, accumulation of mucus and poor clearance of bacteria
- Gradual decrease in lung function further impacted by exacerbations
- Initial colonisation by H. influenzae and Staphylococcus aureus is followed by colonisation by Pseudomonas aeruginosa
- P. aeruginosa predictor of morbidity and mortality
P. aeruginosa’s role in Cystic Fibrosis
- Initial colonisation is by planktonic cells
- Switch to mucoid phenotype and biofilm production associated with poor clearance
- Exopolysaccharide production and anaerobic state associated with antibiotic resistance and inhibition of immune clearance
- P. aeruginosa produces large number of virulence factors many regulated by quorum- sensing control mechanisms
describe Formation of Biofilms by P. aeruginosa
- Attachment facilitated by biofilms = flat
- Initial attachment by flagella, Down-regulation of flagella Siderophore production Type IV pili, twitching motility = structured
what is the structure of the P. aeruginosa Matrix?
- Alginate – acetylated polymer of nonrepetitive monomers of beta-1,4-linked L-guluronic and D- mannuronic acids
- Psl – mannose-rich and galactose-rich polysaccharide
- Pel – glucose-rich polymer DNA and Protein
Functions of Psl and Pel
Initial attachment
Differentiation
Pellicle-type biofilms
explain P. aeruginosa Biofilms in CF
- Initial Colonisation of CF lung by Non-mucoid strains
- Repression of algU by mucABCD
- Progression to Mucoid strains producing alginate (frequent mutation in negative regulator mucA)
Functions of Alginate?
- alginate-antibodies antimicrobial compounds antibiotics
- adsorbed by ROS bactericidal and causing Tissue damage
- IFNg-activated phagocyte
what are Dental Biofilms?
+ Dental biofilms (plaque) is a prerequisite to dental caries
+ Dental biofilms consistent of a heterogenous mix of bacterial species which undergoes temporal changes
+ Metabolism of bacteria in the plaque generates acid leading to removal of the enamel and tooth decay
what are the Temporal Changes in Dental Plaque
- Primary Colonisers
Bind to pellicle (proteins/glycoproteins) First streptococci (S. mutans) Later actinomycetes Synthesize gluten - Bridge Bacteria Fusobacterium Aggregate with primary colonisers
- Late Colonisers Pathogens Porphyromonas ginivalis Bacteriodes forsythus Actinobacillus actinomycetiemcomitans Treponema denticola
whats the structure of Dental Biofilm Matrix?
+ Sucrose primary substrate for Extracellular PolySaccharides (EPS)
+ Synthesized by glucosyl- > fructosyly- transferases
+ S. mutans synthesize alpha(1-3)-linked insoluble glucans, alpha(1-6)-linked soluble glucans alpha(2- 6)-linked fructans
+ Form porous/bulky biofilms facilitating fermentation and acid production as well as adherence
name 3 Biofilms and their Implant Infections
- Endotracheal tube- P. aeruginosa=Pneumonia
- Joint replacement- S. epidermis=Septicaemia
- Prosthetic valve- S.epidermis=Endocarditis
Biofilm Summary
- Biofilms are structured communities that form when planktonic bacteria adhere to host surfaces and secrete exopolysaccharides and/or DNA
- Biofilms facilitate attachment and immune evasion by mediating resistance to phagocytosis, immune effectors and antibiotics
- Biofilms, alginate production and QS-regulated virulence factors contribute to pathogenesis of P. aeruginosa during cystic fibrosis
- Dental biofilms contribute to disease by recruiting pathogenic organisms and changing the biochemical characteristics of bacterial communities
what is Complement Resistance?
+ Resistance to deposition of complement effectors
+ Masking of ligands for complement activators
+ Ineffective complement-activation
+ Recruitment of complement regulators to prevent activation
what is Moraxella catarrhalis?
+ M. catarrhalis is a Gram negative diplococcus
+ Frequent cause of otitis media, sinusitis and lower respiratory tract infections in people with pulmonary problems
+ Common commensal of pharynx of children + Two ubiquitous surface proteins (UspA1 and UspA2) contribute to adherence
how do UspA Proteins Mediate Complement Resistance
+ UspA1 (88-kDa), UspA2 (62-kDa) and UspA2H (92-kDa)
+ UspA1 and UspA2 are 43% identical but share a 140 amino acid domain (93%)
+ UspA1/UspA2 bind C3 and C4b and prevent activation of classical and alternative pathways
+ UspA2 binds vitronectin preventing MAC activation
how do we know M. catarrhalis is Resistant to the Classical and Alternative Complement Pathways?
EGTA inhibits classical pathway (C4b2b)
EDTA inhibits both pathways
Alternative (C3bBb)
what does C4BP do?
Regulates Complement Activation and is recruited by UspA Proteins
how does UspA2 Bind C3 and Inhibit Complement Activation
+ C3, ~1 mg/ml in serum
+ Attaches to bacterial surface in absence of Ab
+ Cleave to release C3b
+ Activates alternative complement pathway
+ C3 bound non- covalently by UspA2 preventing cleavage to C3b
how does Vitronection Inhibits Assembly of MAC?
Vitronectin prevents assembly of MAC by binding to the membrane binding domain of the C5 convertase or by inhibiting polymerisation of C9 stopping pore formation
whats the affect of UspA2 of M. catarrhalis Binding to Vitronectin
prevents MAC assembly imparting serum resistance to M. catarrhalis
how does Factor H Control Complement Activation
C3bBb binds fH inactivating iC3b
how does Neisseria meningitidis Recruit fH?
- A 36-kDa surface-located lipoprotein, fHBP, binds fH and prevents formation of or inactivates the C3 convertase of the alternative pathway. This protein is fully active in encapsulated meningococcal cell.
whats the action of fHBP
- Mediates Serum Resistance
- fHBP and NspA have fH Binding Activity
what are the N. meningitidis Variations in Presence of fHBP and NspA
+ fHBP and NspA prevent deposition of C3 and activation of AP
+ Strains exhibit variations in expression of fHBP and NspA
+ Expression of fHBP controlled by regulator, FNR, resulting in induction of fHBP expression at low oxygen levels
+ Strain-to-strain variations in fHBP due to differences in FNR expression
+ fHBP is a major vaccine candidate
name 3 microbes and their Complement Evasion Strategies
N. gonorrhoeae- BP PorB + PilC - C4BP
N. meningitidis- PorA + Opc - C4BP and Vitronectin
S. pyogenes- M proteins + Fba - C4BP, fH
Complement Resistance Summary
- Bacteria recruit host regulatory proteins and prevent activation of the complement pathways
- Bacteria encode surface proteins for binding of fH, C4BP and vitronectin
- Bacterial pathogens often exhibit multiple ways of inhibiting complement activation
