ABSTRACT
Intestinal Epithelial Cells (IEC) provide the first line of defense against enteric pathogenic bacteria. However
bacterial pathogens developed different strategies to colonize intestinal epithelium causing severe
gastrointestinal disorder. One strategy, used by ‘attaching and effacing’ bacteria (AEB), such as
enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), involves bacterial adhesion to IEC
without entering the host cells. The other strategy used by adherent Invasive E. coli (AIEC) or Salmonella
results in bacterial internalization into IEC. These different colonization routes bear a key mechanistic
similarity: they require rearrangements of the epithelial actin cytoskeleton to enable bacterial attachment and
invasion. One important but understudied mechanism of pathogen-induced cytoskeletal remodeling involves a
major actin cytoskeleton motor, non-muscle myosin II (NM-II). Our preliminary data demonstrate a striking
dualism of NM-II-dependent regulation of bacterial-IEC interactions. Pharmacologic and genetic inhibition of
NM-II increases AEB attachment to IEC, but inhibits AIEC invasion in vitro and in vivo. These contrasting roles
of NM-II in IEC-bacterial interactions likely reflect exploiting two different NM II-dependent functions: its actin
filament contractility and actin filament cross-linking activity, by different pathogens. This exciting data provides
a scientific premise for the following innovative hypothesis: NM-II plays a dual role in regulating enteric
pathogen interactions with intestinal epithelium by inhibiting AEB attachment to epithelial cells, but
promoting epithelial entry of invading pathogenic bacteria.
This hypothesis will be tested in the following Aims: (1) to determine the roles of NM-II in regulating intestinal
epithelial cell interactions with attaching and effacing bacteria; (2) to dissect the mechanisms of NM-II-driven
internalization of invading bacteria into intestinal epithelial cells. We will study EPEC, EHEC, AIEC and
Salmonella colonization of model IEC cell lines, organoid-derived IEC monolayers in vitro and intestinal
colonization by Citrobacter rodentium, AIEC and Salmonella in vivo. NM-II activity will be modulated by
pharmacologic and genetic approaches. The genetic approach will target two major epithelial NM-II isoforms,
NM-IIA and NM-IIC, by selectively downregulating their expression in human IEC using CRISPR/Cas9 gene
editing and RNA interference. In vivo NM-II functions will be examined by using mice with either intestinal
epithelial specific knockout of NM-IIA, or total knockout of NM-IIC. To determine which NM-II activity is
essential for bacterial-IEC interactions, we will utilize IEC cells and transgenic mice expressing NM-IIA mutants
deficient in either actin filament cross-linking, or filament contraction activities. Significance: the proposal will
reveal novel insights into understanding how the intestinal epithelium controls pathogenic bacterial infections
and will identify new targets for therapeutic interventions to treat diseases caused by enteric pathogens.
