Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors

Enteropathogenic and enterohemorrhagic Escherichia
coli cause enteric diseases resulting in significant
morbidity and mortality worldwide. These pathogens remain extracellular
and translocate a set of type III secreted effector proteins into
host cells to promote bacterial virulence. Effectors manipulate host
cell pathways to facilitate infection by interacting with a variety
of host targets, yet the binding partners and mechanism of action
of many effectors remain elusive. We performed a mass spectrometry
screen to identify host targets for a library of effectors. We found
five known effector targets and discovered four novel interactions.
Interestingly, we identified multiple effectors that interacted with
the microtubule associated protein, ensconsin. Using co-immunoprecipitations,
we confirmed that NleB1 and EspL interacted with ensconsin in a region
that corresponded to its microtubule binding domain. Ensconsin is
an essential cofactor of kinesin-1 that is required for intracellular
trafficking, and we demonstrated that intracellular trafficking was
severely disrupted during wild type EPEC infections but not during
infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics
for identifying effector–host protein interactions and suggest
that vesicular trafficking is a crucial cellular process that may
be targeted by NleB1 and EspL through their interaction with ensconsin.