Bacterial Biofilms: Synthesis, Structure and Applica8ons Bryan W. Berger Department of Chemical and Biomolecular Engineering Program in Bioengineering Bad Bugs, No Drugs – 10 New An8bio8cs by 2020 (IDSA) hp://www.birkocorp.com Bacteria have evolved numerous anbioc resistance mechanisms 3. Biofilms • Mo6le bacteria adhere to surface • Secrete extracellular polymeric substance (EPS) • Comprised of polysaccharides, DNA, and protein • Acts as diffusion barrier against an6bio6cs Planktonic bacteria EPS Mucoid bacteria A"achment Growth Bacteria encased in EPS are protected from anmicrobials as well as other stress factors including phagocytosis and dehydraon An6microbial Planktonic bacteria EPS Mucoid bacteria A"achment Growth cdc.gov S. maltophilia: An Important, Emerging, Nosocomial Pathogen – Emily Wong MD, PhD (Lehigh Valley Hospital) Emerging Infecous Disease, Volume 8, Number 9—September 2002 Stenotrophomonas maltophilia is an intrinsic mul8drug resistant (MDR), nosocomial pathogen of growing concern • Third most common nosocomial non-fermen6ng Gran-negave bacilli • Third most common cause of late-onset ven6lator-acquired pneumonia • Second most common bacteria isolated from lungs of CF paents • Associated with high mortality rate (30%) with few treatment op6ons due to intrinsic MDR • Capable of adhering to and producing biofilm on IB3-1 bronchial cells as well as numerous inert surfaces found in indwelling medical devices such as catheters and ven6lators • Biofilm formaon in linked to virulence and MDR Number of Percentage of Class Name Resistant Strains Resistant Strains (n=28) (%) β-lactam Ampicillin 28 100 Streptomycin 28 100 Specnomycin 28 100 Aminoglycoside Kanamycin 28 100 Gentamicin 27 96.4 Glycopepde Zeocin 28 100 Nalidixic acid 7 25 Broad-spectrum Tetracyline 5 17.9 Chloramphenicol 3 10.7 An6bio6c screening of S. maltophilia clinical strains received from Biofilm producing Stenotrophomonas maltophilia Lehigh Valley Hospital Strain BB2 collected from Lehigh Valley Hospital S. maltophilia Clustering and Biofilm Forma8on in Culture BB6 R551-3 Celluronic acid: Primary component of plant cell wall Alginic acid: Component of bacterial biofilm Hyaluronic acid: Component of mammalian ECM Other poylsaccharides: pectic acid, mucin, acetylated alginate, heparin Many biological important polysaccharides contain uronic acids • Hydroxyl group of C6 is oxidized to form carboxylic acid • Results in anionic polysaccharide Oxidaon • Four major groups 1. Alginates, three block types • Poly-β-D-mannuronic acid (polyManA) • Poly-α-L-guluronic acid (polyGulA) • Alternang ManA and GulA blocks (polyMG) ManA GulA Many biological important polysaccharides contain uronic acids • Hydroxyl group of C6 is oxidized to form carboxylic acid • Results in anionic polysaccharide Oxidaon • Four major groups 1. Alginates, three block types • Poly-β-D-mannuronic acid (polyManA) • Poly-α-L-guluronic acid (polyGulA) • Alternang ManA and GulA blocks (polyMG) C5 epimers ManA GulA Many biological important polysaccharides contain uronic acids • Four major groups 1. Alginates, three block types • Poly-β-D-mannuronic acid (polyManA) • Poly-α-L-guluronic acid (polyGulA) • Alternang ManA and GulA blocks (polyMG) ManA ManA ManA ManA Banin, E. et al. “Iron and Pseudomonas aeruginosa biofilm formaon.” PNAS. 102(31):11076-11081 Many biological important polysaccharides contain uronic acids • Four major groups 1. Alginates, three block types • Poly-β-D-mannuronic acid (polyManA) • Poly-α-L-guluronic acid (polyGulA) • Alternang ManA and GulA blocks (polyMG) GulA GulA GulA GulA GulA Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Many biological important polysaccharides contain uronic acids • Four major groups 1. Alginates, three block types • Poly-β-D-mannuronic acid (polyManA) • Poly-α-L-guluronic acid (polyGulA) • Alternang ManA and GulA blocks (polyMG) ManA GulA ManA GulA Many biological important polysaccharides contain uronic acids • Four major groups 3. Glucuronans (poly-β-D-glucuronic acid, polyGlcA) o Major component of cell well in green algae o Component of several bacterial exopolysaccharides GlcA GlcA GlcA GlcA GlcA Many biological important polysaccharides contain uronic acids • Hyaluronic acid (HA) • Major component of extracellular matrix in nearly all connec6ve 6ssue • Repeang disaccharide of GlcA and N-acetylglucosamine (GlcNAc) • Degree of polymerizaon up to 25,000 disaccharide units (MW = 10 MDa) • High viscosity acts as defense mechanism against infec6ous agents and secreted toxins by preven6ng diffusion into deep 6ssue GlcA GlcNAc GlcA GlcNAc Polysaccharide lyases (PLs) catalyze the cleavage of uronic acid containing polysaccharides • Enzymes which cleave O-glycosidic bond between sugar rings • Classified into 21 polysaccharide lyase families (PL families) based on secondary structure fold and substrate specificity • Reac6on proceeds via a β-eliminaon mechanism, resul6ng in the formaon of an double bond at the new non-reducing end Pseudomonas aeruginosa expresses a periplasmic alginate lyase (AlgL) which regulates the chain length of secreted alginate and prevents lethal build-up of alginate in periplasm Jain, S. et al. “Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa.” Infec6on and Immunity. 2005. 73(10):6429-6436. Pseudomonas aeruginosa expresses a periplasmic alginate lyase (AlgL) which regulates the chain length of secreted alginate and prevents lethal build-up of alginate in periplasm Jain, S. et al. “Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa.” Infec6on and Immunity. 2005. 73(10):6429-6436. Pseudomonas aeruginosa expresses a periplasmic alginate lyase (AlgL) which regulates the chain length of secreted alginate and prevents lethal build-up of alginate in periplasm Jain, S. et al. “Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa.” Infec6on and Immunity. 2005. 73(10):6429-6436. Genomic sequencing of clinical isolate Stenotrophomonas maltophilia K279a revealed a putave alginate lyase (Smlt1473) absent from the genomes of environmental strains • Strain K279a isolated from blood of cancer paent who developed an infec6on which did not respond to an6bio6c treatment • Smlt1473 restricted to a subset of S. maltophilia clinical isolates and other related pathogens such as Achromobacter and Bordetella (≥ 60% amino acid sequence iden6ty) • Predicted to belong to PL-5 family • No evidence to suggest alginate is a major component of S. maltophilia biofilm Goals: • Characterize unique, restricted lyase • Determine substrate specificity • Postulate possible biological role of lyase BB40, 43, Negave control BB8-13 BB1-7 BB16-18, 20-23 BB24-30 BB32-38 Posi6ve band (533 bp) gDNA sreen of smlt1473 Expected size: 533 bp Number of strains screened: 36 Number of posi6ve strains: 28 Percent posi6ve: 77.8% Gels run from the top down with ladder loaded last (i.e. gel #1 is BB1, BB2, BB3, BB4, BB5, BB6, BB7, Ladder) Smlt1473 was heterologously expressed in E. coli and purified in a one step manner via immobilized metal ion affinity chromatography 1 pET28a-smlt1473 pET28a Low pET28a-smlt1473 0.9 WC Low Mid High WC Low Mid High pET28a 0.8 75 0.7 50 0.6 0.5 37 0.4 Mid High 0.3 25 Absorbance at280 nm 0.2 20 0.1 15 0 0 20 40 60 80 100 120 140 160 10 A Time (minutes) C 1.6 Hyaluronic Acid (pH=5) 1.4 polyGlcA (pH=7) polyManA (pH=9) 1.2 1 0.8 0.6 0.4 Absorbance at560 nm 0.2 0 Low Mid High Low Mid High B pET28a-smlt1473 pET28a D Heterologous secreon of Smlt1473 is dependent on a predicted N-terminal lipoprotein signal WT C23F Cyto Peri Cyto Peri 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa 15 kDa Heterologous expression of Smlt1473 resulted in extracellular lyase acvity polyGlcA polyManA WT C23F Forma8on of unsaturated products allows for the monitoring of enzyma8c ac8vity by measuring absorbance at 235 nm with respect to me 0.7 0.6 0.5 0.4 0.3 Absorbance @ 235 nm 0.2 0.1 0 0 20 40 60 80 100 120 Time (seconds) ) 50 Acetate Substrate specificity analysis revealed -1 Phosphate Hyaluronic Acid mg Tris a pH-dependence on lyase ac8vity -1 40 • Maximum ac6vity against min 30 • Hyaluronic Acid at pH 5 235 nm A Δ • PolyGlcA at pH 7 20 • Alginate based substrates at pH 9 10 0 Specific Activity( Specific 3 4 5 6 7 8 9 10 11 pH ) 900 ) 80 Acetate Acetate -1 Phosphate polyGlcA -1 Alginate 800 70 Phosphate mg Tris mg Tris -1 -1 700 Glycine 60 Glycine min 600 min 50 235 nm 500 235 nm A A 40 Δ 400 Δ 30 300 200 20 100 10 0 0 Specific Activity( Specific Activity( Specific 3 4 5 6 7 8 9 10 11 3 4 5 6 7 8 9 10 11 pH pH Smlt1473 was found to be ac8ve against a wide variety of uronic acid containing polysaccharides Substrate pH Specific Ac8vity (U/mg) Alginate 9 20.4 ± 0.7 Poly-β-D-mannuronic acid (polyManA) 9 68.5 ± 2.9 Poly-α-L-glucuronic acid (polyGulA) 9 2.1 ± 0.2 Alternang ManA/GulA (polyMG) 9 12.8 ± 0.4 Poly-β-D-glucuronic acid (polyGlcA) 7 848.3 ± 6.3 Poly-α-D-galacturonic acid (polyGalA) 5,7,9 ND Hyaluronic Acid (HA) 5 42.3 ± 1.3 Mucin 5,7,9 ND Heparin 5,7,9 ND No detectable ac6vity (detec6on limit: 0.001 absorbance units at 235 nm per minute). Smlt1473 was found to be ac8ve against a wide variety of uronic acid containing polysaccharides Substrate pH Specific Ac8vity (U/mg) Alginate 9 20.4 ± 0.7 Poly-β-D-mannuronic acid (polyManA) 9 68.5 ± 2.9 Poly-α-L-glucuronic acid (polyGulA) 9 2.1 ± 0.2 Alternang ManA/GulA (polyMG) 9 12.8 ± 0.4 Poly-β-D-glucuronic acid (polyGlcA) 7 848.3 ± 6.3 Poly-α-D-galacturonic acid (polyGalA) 5,7,9 ND Hyaluronic Acid (HA) 5 42.3 ± 1.3 Mucin 5,7,9 ND Heparin 5,7,9 ND No detectable ac6vity (detec6on limit: 0.001 absorbance units at 235 nm per minute). Analysis of oligosaccharide products revealed an endolyc cleavage Hyaluronic Acid, pH 5 Δ-GlcNAc-GlcA-GlcNAc Δ-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc Δ-GlcNAc-(GlcA-GlcNAc) Δ-GlcNAc 3 polyGlcA, pH 7 Δ-GlcA Δ-GlcA-GlcA Δ-GlcA-GlcA-GlcA Δ-ManA-ManA polyManA, pH 9 Δ-ManA Δ-ManA-ManA-ManA Δ-(ManA)4 Δ-(ManA)5 Celluronic acid: Primary component of plant cell wall Alginic acid: Hyaluronic acid: Other poylsaccharides: pecc acid, mucin, acetylated alginate, heparin β-eliminaon mechanism proceeds in three steps 1.