posted on 2021-04-14, 19:36authored byJoshua
E. Petch, Pratik Gurnani, Gokhan Yilmaz, Francesca Mastrotto, Cameron Alexander, Stephan Heeb, Miguel Cámara, Giuseppe Mantovani
The
selective isolation of bacteria from mixed populations has
been investigated in varied applications ranging from differential
pathogen identification in medical diagnostics and food safety to
the monitoring of microbial stress dynamics in industrial bioreactors.
Selective isolation techniques are generally limited to the confinement
of small populations in defined locations, may be unable to target
specific bacteria, or rely on immunomagnetic separation, which is
not universally applicable. In this proof-of-concept work, we describe
a novel strategy combining inducible bacterial lectin expression with
magnetic glyconanoparticles (MGNPs) as a platform technology to enable
selective bacterial isolation from cocultures. An inducible mutant
of the type 1 fimbriae, displaying the mannose-specific lectin FimH,
was constructed in Escherichia coli allowing for “on-demand” glycan-binding protein presentation
following external chemical stimulation. Binding to glycopolymers
was only observed upon fimbrial induction and was specific for mannosylated
materials. A library of MGNPs was produced via the grafting of well-defined
catechol-terminal glycopolymers prepared by reversible addition–fragmentation
chain transfer (RAFT) polymerization to magnetic nanoparticles. Thermal
analysis revealed high functionalization (≥85% polymer by weight).
Delivery of MGNPs to cocultures of fluorescently labeled bacteria
followed by magnetic extraction resulted in efficient depletion of
type 1 fimbriated target cells from wild-type or afimbriate E. coli. Extraction efficiency was found to be dependent
on the molecular weight of the glycopolymers utilized to engineer
the nanoparticles, with MGNPs decorated with shorter Dopa-(ManAA)50 mannosylated glycopolymers found to perform better than
those assembled from a longer Dopa-(ManAA)200 analogue.
The extraction efficiency of fimbriated E. coli was also improved when the counterpart strain did not harbor the
genetic apparatus for the expression of the type 1 fimbriae. Overall,
this work suggests that the modulation of the genetic apparatus encoding
bacterial surface-associated lectins coupled with capture through
MGNPs could be a versatile tool for the extraction of bacteria from
mixed populations.