Enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes generate actin‐rich structures that are used for their colonization of host cells. EPEC remains extracellular, attaches to the host plasma membrane and secretes a variety of effector proteins that are used to control the host cells. The most dramatic morphological phenotype generated during these infections is the formation of actin‐rich protrusions called pedestals that are needed for E. coli‐based disease. L. monocytogenes enters its host cell and once in the cytosol it generates a bacterial protein (called ActA) that recruits actin polymerizing proteins to 1 pole of the microbe forming a comet tail that is used for movement within and amongst the host cells Previously, our lab conducted a mass spectrometry analysis of concentrated EPEC pedestals and over 90 novel proteins were identified. From this list, we selected a subset for confirmatory analysis and to determine whether their presence required the pre‐formation of the actin‐rich structures. These proteins included a calponin protein (CNN), dihydropyrimidinase‐like protein (CRMP4), nucleoside kinase (NK), mitogen‐activated protein pathway kinase (MK), and a ubiquitin‐conjugating enzyme (Ube2N). Because these novel proteins were observed in EPEC pedestals, we hypothesized that these five protein candidates are important for EPEC pedestals and L. monocytogenes comet tails. To study this, we first confirmed that these proteins were enriched in EPEC pedestals by immunolocalizing the proteins in EPEC‐infected cells. CNN, and NK were found throughout the full length of the pedestal while CRMP4, MK, and Ube2N were concentrated at the apical tip of EPEC pedestals. In L. monocytogenes‐infected cells, CNN and NK were found in actin clouds, comet tails, and listeriopods. CRMP4 immunolocalized only at invasion sites and listeriopods while MK was only present at listeriopods. To ensure that recruitment of these proteins were independent of bacterial attachment on the host cell and required formation of the actin‐rich structures, we infected cells with either a tir‐deficient EPEC mutant or an actA‐deficient L. monocytogenes mutant as both mutants cannot generate actin‐structures. None of the five proteins were recruited to the mutants suggesting that their presences are dependent on the formation of the actin‐rich structures. From these results, we have identified novel proteins that are important for the various stages of EPEC and L. monocytogenes infections. By identifying these proteins, we have begun to elucidate the complex web of proteins involved in the formation and maintenance of actin structures during these infections.Support or Funding InformationThis study was funded through NSERC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.