1. y sus aplicaciones ambientales
Sistemas de secreción
y sus aplicaciones ambientales

2. Structure of gram-negative cell wall

3. The envelope of a Gram negative bacterium

4. 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

5. 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

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

7. 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).

8. 1- Surface Display of proteins Engineering bacterial consortia
using the autotransporter domain of Neisseria gonorrhoeae IgA protease:
Engineering bacterial consortia

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

10. Leucine zippers as dimerization domains

11. Neisseria gonorrhoeae IgA protease
Periplasm
Cytoplasm OM IM

12. The b barrel structure of OM proteins

13. 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

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

15. Cell aggregation studies
Fosb
Junb
Fosb+ Junb

16. Floculation in liquid culture
Fosb
Junb
Fosb+ Junb

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

18. 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

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

20. HOST PASSIVE IMMUNIZATION
viruses
neutralizing Ab
BACTERIA
HOST EPITHELIUM

21. The structure of antibodies and scFvs

22. E. Coli haemolysin translocator

23. Crystal structure of TolC

24. TolC has a 3.5 nm-diameter internal cavity

25. Secretion of scFvs using the E. coli hly system

26. Secretion of scFvs using the E. coli hly system

27. ScFvs-secreted by the Hly system are active

28. TGEV neutralization in vitro by
E. coli secreted scFv

29. 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

30. 3- Engineering E. coliI type I fimbriae:
Redirecting bacterial adhesion

31. DIRECTED CELL ADHESION
EUKARYOTIC CELL
BACTERIA A
BACTERIA B
Inorganic surface

32. Escherichia coli Type I fimbriae

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

34. 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