y sus aplicaciones ambientales Sistemas de secreción y sus aplicaciones ambientales

Structure of gram-negative cell wall

The envelope of a Gram negative bacterium

The biological roles of the bacterial secretion systems Adhesion: fimbriae, OM adhesins Cell motility: flagella Horizontal gene transfer: conjugative pili Colonization: toxins, colicins, proteases Virulence: type III secretion systems Bacterial resistance: antibiotics, organics, heavy-metals

Gram negative secretion systems Special Machinery Heterologous secretion Name Example Signals Function E. coli Haemolysin Type I C-end 3 Toxins (RTX) Yes K. oxytoca Pullulanase Type II Enzymes Difficult N- C-ends > 12 Y. enterocolitica Yops N- and mRNA > 14 Inject proteins Difficult Type III Inject proteins and DNA A. tumefaciens VirB Difficult Type IV N- ? > 10 Proteases, Toxins, Adhesins ATs N. gonorroheae IgA protease Yes N- C-ends 1

Bacteria Bacterial secretion/display of heterologous proteins Sec OMPs Fimbriae Bacteria

Bacterial secretion and display of proteins OMPs (LamB), Lpp-ompA hybrids, Ice-nucleation protein, ATs. Vector systems: Pili and flagella. Secretion systems: type I Applications: Live vaccines. Bioremediation (pesticides, heavy metals). Altered adherence (biofilms, targeting).

1- Surface Display of proteins Engineering bacterial consortia using the autotransporter domain of Neisseria gonorrhoeae IgA protease: Engineering bacterial consortia

Can we make an artificial bacterial consortium? Bacterium A Interaction domain OM anchor Bacterium B

Leucine zippers as dimerization domains

Neisseria gonorrhoeae IgA protease Periplasm Cytoplasm OM IM

The b barrel structure of OM proteins

Expression of leucine zippers on the surface of E. coli Fosb Junb kDa Plac SP 200 igAb 97 66 L L L L L 31

Expression of leucine zippers on the surface of E. coli Control Fosb Junb

Cell aggregation studies Fosb Junb Fosb+ Junb

Floculation in liquid culture Fosb Junb Fosb+ Junb

Virus neutralisation with single-chain antibodies 2- Secretion of active proteins using the E. coli haemolysin transporter system: Virus neutralisation with single-chain antibodies

IMPORTANCE OF MUCOSAL IMMUNITY The mucosal body surface represents ~ 400 m2 vs ~1.8m2 of skin Over 90% infections are initiated at the mucosa There is a specialized mucosa-associated lymphoid tissue (MALT) Attenuated mucosal pathogens can be used to target MALT

HOST EPITHELIUM The gut epithelium as the port of entry of TGEV viruses viruses viruses HOST EPITHELIUM

HOST PASSIVE IMMUNIZATION viruses neutralizing Ab BACTERIA HOST EPITHELIUM

The structure of antibodies and scFvs

E. Coli haemolysin translocator

Crystal structure of TolC

TolC has a 3.5 nm-diameter internal cavity

Secretion of scFvs using the E. coli hly system

Secretion of scFvs using the E. coli hly system

ScFvs-secreted by the Hly system are active

TGEV neutralization in vitro by E. coli secreted scFv

Conclusions Secreted scFv-HlyA is oxidized and active The scFv domain does not appear to be oxidized spontaneously Neither Dsbs nor Trxs are involved in oxidation of scFv-HlyA Intracellular scFv-HlyA is in a reduced form Intracellular scFv-HlyA is rapidly degraded unless secreted TrxB mutation inhibits secretion of scFv-HlyA by an unknown mechanism

3- Engineering E. coliI type I fimbriae: Redirecting bacterial adhesion

DIRECTED CELL ADHESION EUKARYOTIC CELL BACTERIA A BACTERIA B Inorganic surface

Escherichia coli Type I fimbriae

Agglutination of yeast cells HB2151 HB101(pFH35) HB101 (pFH35, pPKL115) + mannose

Eukaryotic cell adhesion assay: Probing FimH binding specificity Eukaryotic cells (NRK or HeLa) -Innoculation with E. coli transformed with different plasmids -Incubation at 37ºC for 2 h -Washes -Only fimbriated bacteria displaying adhesin will adhere to eukaryotic cells