Myosin IIA motor regulates attaching-effacing bacteria interactions with intestinal epithelium

Abstract Attaching effacing (A/E) bacteria, such as Enteropathogenic E. coli (EPEC) and Citrobacter rodentium, colonize intestinal epithelial cells (IECs) by inducing remodeling of the epithelial cytoskeleton and formation of prominent actin pedestals at bacterial attachment sites. While non-muscle myosin II (NM II) is a key regulator of the actin cytoskeleton, whether it regulates IEC colonization by A/E pathogens is not known. To address this question, we targeted NM IIA and NM IIC, the NM II paralogs expressed in IECs. Our in vivo studies utilized mouse models with either intestinal epithelial-specific deletion of NM IIA (NM IIA cKO mice), expression of a NM IIA motor domain mutant, or total deletion of NM IIC (NM IIC tKO mice). In vitro experiments utilized IECs (HT-29cF8 and Caco-2BBE) with CRISPR-Cas9-mediated deletion of NM IIA or NM IIC. In addition, NM II activity in vitro was modulated pharmacologically, using either the pan-myosin inhibitor, blebbistatin, or a specific NM IIC activator, 4-hydroxyacetophenone (4-HAP). NM IIA cKO and NM IIA mutant mice demonstrated higher C. rodentium colonization along with more severe mucosal inflammation and colonic crypt hyperplasia as compared to their controls. By contrast, NM IIC tKO mice was indistinguishable from their control with regard to C. rodentium colonization. Blebbistatin treatment increased EPEC attachment to IECs monolayers, whereas 4-HAP did not affect bacterial attachment. Genetic knockout of NM IIA, but not NM IIC, increased EPEC adhesion to IEC monolayers. Importantly, the increase in EPEC attachment exhibited by NM IIA-deficient IECs required intact bacterial Type 3 secretion system and functional Tir effector, indicating that NM IIA functions in actin pedestal assembly. In summary, we describe a novel role for NM IIA in limiting intestinal epithelial colonization by A/E pathogens via inhibition of pathogen-induced remodeling of the actin cytoskeleton. Competing Interest Statement The authors have declared no competing interest. Footnotes This version was of the manuscript has been revised to correct/unify graph labeling in several figures and supplementary figures. List of abbreviations - A/E - attaching/effacing - cKO - conditional knockout - EPEC - Enteropathogenic Escherichia coli - IECs - intestinal epithelial cells - NM II - non-muscle myosin II - T3SS - Type III secretion system - tKO - total knockout