Luis Angel FERNANDEZ - Engineering synthetic adhesins and injectisomes in Escherichia coli K-12...
Channel: Institut des Hautes Études Scientifiques (IHÉS)
Date: 2015-12-21

Engineering synthetic adhesins and injectisomes in Escherichia coli K-12 to target mammalian cells for biomedical applications

One  of  the  aims  of  synthetic  biology  is  the  design  of microorganisms  with  novel capabilities that could be applied
for the development of new vaccines, diagnostic sensors, and  therapeutic  interventions  for  major  diseases  such  as  cancer.  This  presentation  will report the development of two important tools that enable us to precisely program E. coli K-12 bacteria  to:  1)  adhere to  specific target cells;  and  2)  assemble filamentous 
injectisomes from  type  III  secretion  systems (T3SS) 
that  act  as "molecular  syringes"  for translocation  of  specific  proteins  into  mammalian  cells. 
Firstly,  we  designed  synthetic adhesins based on the display of VHH domains on the bacterial cell surface with an outer membrane β-domain  derived  from  intimin  of enterohemorrhagic 
E.  coli (EHEC).  We generated synthetic adhesins against different antigen targets expressed on the surface of mammaliam cells and
have demonstrated the specific adhesion of the engineered 
E. coli bacteria  to  the  target  mammalian  cells using in  vitro
and in  vivo models. Using mouse xenotransplants  of  human  tumor  cell  lines expressing  a  target  antigen,  we  demostrated 
that engineered E.  coli bacteria  colonize  these  tumors  more  efficiently at  lower  bacterial doses (Piñero-Lambea et al., 2015).
Secondly, in order to express functional injectisomes in  a  non-
pathogenic  commensal E.  coli strain  (K-12),  we  reformatted  the 
operons encoding the structural proteins and chaperones needed for the assembly of filamentous injectisomes from enteropathogenic E. coli (EPEC). Our synthetic operon constructs
lack secreted effectors and regulatory elements found in EPEC. Five synthetic operons (sLEE1, sLEE2,  sLEE3,  sLEE4  and  escD) 
were integrated  into  different  sites  of  the  chromosome of E.  coli K-12  under  the  control  of  an  inducible  promoter  (Ptac)  using  a  marker-less strategy.  We  demonstrated that  the  resulting  strain,  named  Synthetic Injector E.  coli (SIEC), assembles  functional injectisomes  upon  induction  with  IPTG  able to  translocate proteins into HeLa cells (Ruano-Gallego et al., 2015). 
Collectively, these results open the possibility  to target 
specific mammalian  cells  with  engineered E.  coli bacteria  and  inject heterologous  proteins of  interest,  such  as antibody  fragments,  immunogens,  enzymes, transcription factors, 
or toxins.
References
-Piñero-Lambea, C., Bodelón, G., Fernández-Periáñez, R., Cuesta, A.M., Álvarez-Vallina, L.,  and  Fernández,  L.Á.  (2015). Programming  Controlled  Adhesion  of E.  coli to  Target 
Surfaces, Cells, and Tumors with Synthetic Adhesins. ACS synthetic biology 4, 463-473.

- Ruano-Gallego, D., Álvarez, B., and Fernández, L.Á. (2015). Engineering the Controlled Assembly  of  Filamentous Injectisomes  in E.  coli K-12  for  Protein  Translocation  into 
Mammalian Cells. ACS synthetic biology 4, 1030-1041.