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Making Attachments: Curli Production in Bacterial Communities Deborah Yager Stanford IISME 2010 Lynette Cegelski, Mentor Funded by Terman Fellowship Award Figure 1C4: MC4100 E.Coli deep-etched on glass before rotary shadowing with Pt showing curli fibrils Outline What is a biofilm? Biofilms and everyday life Curli Basic model for curli protein interactions More details on each protein subunit Congo red binding assay Experimental design What is a Biofilm? Biofilms and the Environment Biofilm Life Cycle The biofilm life cycle in three steps: attachment, growth of colonies (development), and periodic detachment of planktonic cells. Bioflims and Disease Common sites of biofilm infection. Once a biofilm reaches the bloodstream it can spread to any moist surface of the human body. Microbes that colonize vaginal tissue and tampon fibers can become pathogenic, causing inflammation and disease such as Toxic Shock Syndrome. Bioflims and Disease Why Bacteria Make Attachments Bacteria prefer to attach to surfaces rather than live in suspension Advantages to attachment: 1) higher nutrient concentration (carbon source) 2) protection from hostile environments 3) formation of biofilms, “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface.” Advantages of Biofilms In a biofilm, energy required to find food is shared Biofilm members on the edge are first line of defense Bacteria in biofilms secrete a protective matrix of polysaccharides and proteins (i.e. cellulose & pili) One set of secreted proteins form curli fibers Biofilm in the gut Curli as Amyloid Amyloid is an extracellular structure built of protein subunits in a cross-beta sheet formation Cross beta sheet Amyloid aggregates are implicated in several diseases, including Alzheimer’s and Parkinson’s Curli are adhesive extracellular functional amyloid fibers secreted by bacteria Curli Production Curli are secreted by Gram negative bacteria including Escherichia Coli (E. coli) and Salmonella. Together with cellulose and other extracellular matrix components, curli help bacteria attach to surfaces and stick together to form a biofilm. While curli form spontaneously in vitro (in a test tube with no cells), in vivo (in life) curli production requires specialized molecular machinery. Curli Molecular Machinery Genetics Nomenclature csgA means “gene that codes for protein A” CsgA means “Protein A coded by curli specific gene (csg)” Practice: What does CsgG mean? What does csgG mean? Quick Guide to Curli Proteins CsgA: major curli subunit, forms fibers CsgB: minor curli subunit, nucleation CsgD: transcriptional activator CsgE: gatekeeper or plug CsgF: chaperone protein CsgG: outer membrane pore CsgA Major fiber subunit 13 kDa w/o Sec sequence Amyloidogenic imperfect repeating units (R1-5) Gln/Asn-rich repeat motif 3 domains • • • N-terminal Sec sequence (2 kDa) First 22 amino acids C-terminal Amyloid core Fibers rich in -sheet secondary structure CsgA (cont.) Polymerization responds to self-seeding and CsgB nucleation Proposed model: R1 interacts with CsgB, R5 interacts with R1 of next CsgA A A A A B B F E Congo Red Binding Assay Congo red is an amyloid dye that binds to CsgA subunits when they are polymerized in a fiber Bacterial cells grown on nutritional media with Congo red will stain red if curli fibers are present CR Congo Red Binding Assay Curli - Curli + Interbacterial Complementation Interbacterial Complementation Interbacterial Complementation (IC): • recipient strain is streaked first (down) • donor strain streaked next (across) Congo red dye binds to polymerized CsgA ∆A mutant = no fibers, cells white on CR plate • Figure 3D4: Donor CsgA+ CsgB-, Recipient CsgA- CsgB+ Can accept CsgA in IC CsgB Figure 1A2 Minor fiber subunit 17 kDa (whole) 5 repeating units Gln/Asn-rich repeat motif ~30% sequence identity with CsgA R1-R4 amyloidogenic A CsgB (cont.) A A B B A F E Nucleates CsgA WT CsgB cell-associated ∆B mutant = no fibers, cells white on CR plate • • Secretes soluble CsgA outside the cell Can donate CsgA in IC CsgD 5 kDa (whole) Transcriptional regulator for curli genes Critical activator of cellulose biosynthesis pathway ∆D mutant = no curli or cellulose Represses genes inhibiting biofilm formation in E. coli CsgE A A A A B B F E 15 kDa (whole) Interacts with CsgG at outer membrane Gatekeeper for CsgG pore ∆E mutant: very few fibers (pale cells, CR-), arrange into rings • • • Figure 3D4: Donor CsgA+ CsgB-, Acceptor CsgA- CsgB+ Cannot donate CsgA Can accept and form fibers Suggests CsgE needed for CsgA polymerization stability CsgF A A A A B B F E Chaperone-like, helps CsgB nucleation 15 kDa (whole) Secreted across outer membrane, interacts with CsgG Degraded with increasing concentration of proteinase K, suggests CsgF exposed on cell surface • Same observed in ∆A, ∆B, ∆E mutants, localization not dependent on A, B, or E CsgF (cont.) • ∆F mutant = no cellassociated fibers, CsgA secreted soluble • CR: Scraped whole cells appear white, suggests fibers mislocalized to agar Figure 3A/B5 CsgG A A A A B B Outer membrane pore • F E • 30.5 kDa (whole) ∆G mutant = no fibers • • Figure 1A6 Stabilizes curli subunit proteins 12-15 nm across with 2 nm pore CsgA and CsgB not secreted to cell surface, not in periplasmic space Can neither donate nor accept in IC Congo Red Binding Assay Curli - Curli + Experimental Design Single variable of interest: want to minimize influence of other variables Controlled experiment uses scientific controls: positive control and negative control Positive control confirms that your methods can produce a positive result • minimizes false negatives Negative control confirms that your methods do not produce an unrelated effect • • minimizes false positives demonstrates baseline result obtained without positive result (“background” value) Our Experiment Experimental variable: a single curli-specific gene (csg) mutation (MC4100DcsgA) Positive control: wild type MC4100 cells • shows a positive result with our methods (Congo red binding) Negative control: cells missing complete csg sequence (MC4100Dcsg) • shows “background” (negative) result obtained when no curli genes are expressed Curli mutant UTI89 UTI89 MC4100 References 1. Cegelski Lab. “Structure, Function, and Disruption of Microbial Amyloid Assembly and Biofilm Formation.” 2009. 2. Hammer, N.D.; Schmidt, J.C.; Chapman, M.R. The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization. PNAS 2007, 104, 12494-12499. 3. Cegelski, L.; Pinkner, J.S.; Hammer, N.D.; Cusumano, C.K.; Hung, C.S.; Chorell, E.; Aberg, V.; Walker, J.N.; Seed, P.C.; Almqvist, F.; Chapman, M.R.; Hultgren, S.J. Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation. Nature Chemical Biology 2009, 12, 913-919. 4. Chapman, M.R.; Robinson, L.S.; Pinkner, J.S.; Roth, R.; Heuser, J.; Hammar, M.; Normark, S.; Hultgren, S.J. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 2002, 295, 851855. 5. Nenninger, A.A.; Robinson, L.S.; Hultgren, S.J. Localized and efficient curli nucleation requires the chaperone-like amyloid assembly protein CsgF. PNAS 2009, 106, 900-905. 6. Robinson, L.S.; Ashman, E.M.; Hultgren, S.J.; Chapman, M.R. Secretion of curli fibre subunits is mediated by the outer membrane-localized CsgG protein. Molecular Microbiology 2006, 59, 870-881. 7. Wang, X.; Chapman, M.R. Curli provide the template for understanding controlled amyloid propagation. Prion 2008, 2, 57-60. 8. Wang, X.; Zhou, W.; Ren, J.; Hammer, N.D.; Chapman, M.R. Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis. PNAS 2010, 107, 163-168. 9. Ashman, E.; Chapman, M.R. Polymerizing the fibre between bacteria and host cells: the biogenesis of functional amyloid fibres. Cellular Microbiology 2008, 1-8. 10. Zakikhany, K.; Harrington, C.R.; Nimtz, M.; Hinton, J.C.D.; Romling, U. Unphosphorylated CsgD controls biofilm formation in Salmonella enterica serovar Typhimurium. Molecular Microbiology 2010, accepted article. 11. Ashman Epstein, A.; Reizian, M.A.; Chapman, M.R. Spatial clustering of the curlin secretion lipoprotein requires curli fiber assembly. Journal of Bacteriology 2009, 191, 608-615.