Enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium, and Listeria monocytogenes usurp the actin cytoskeleton for their attachment, internalization and transport within and amongst infected cells. To try to gain a greater understanding of the molecular components utilized by these microbes during their infections we previously concentrated actin‐rich structures generated during EPEC infections (called pedestals) and identified the heat shock cognate 70 protein (Hsc70) as a potential candidate. This multifunctional protein classically acts as a chaperone for the proper folding of a variety of proteins and is involved in uncoating clathrin from coated pits. Here we demonstrated that Hsc70 is recruited to actin structures generated during EPEC, Listeria and Salmonella infections, but not to the same location as clathrin. Anat Rec, 301:2095–2102, 2018. © 2018 Wiley Periodicals, Inc.

The ingestion of enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, or Salmonella enterica serovar Typhimurium leads to their colonization of the intestinal lumen, which ultimately causes an array of ailments ranging from diarrhea to bacteremia. Once in the intestines, these microbes generate various actin‐rich structures to attach, invade, or move within the host intestinal epithelial cells. Although an assortment of actin‐associated proteins has been identified to varying degrees at these structures, the localization of many actin stabilizing proteins have yet to be analyzed. Here, we examined the recruitment of the actin‐associated proteins, calponin 1 and 2 at EPEC pedestals, L. monocytogenes actin clouds, comet tails and listeriopods, and S. Typhimurium membrane ruffles. In other systems, calponins are known to bind to and stabilize actin filaments. In EPEC pedestals, calponin 1 was recruited uniformly throughout the structures while calponin 2 was enriched at the apical tip. During L. monocytogenes infections, calponin 1 was found through all the actin‐rich structures generated by the bacteria, while calponin 2 was only present within actin‐rich structures formed by L. monocytogenes near the host cell membrane. Finally, both calponins were found within S. Typhimurium‐generated membrane ruffles. Taken together, we have shown that although calponin 1 is recruited to actin‐rich structures formed by the three bacteria, calponin 2 is specifically recruited to only membrane‐bound actin‐rich structures formed by the bacteria. Thus, our findings suggest that calponin 2 is a novel marker for membrane‐bound actin structures formed by pathogenic bacteria. Anat Rec, 301:2103–2111, 2018. © 2018 Wiley Periodicals, Inc.

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.

Listeria monocytogenes and enteropathogenic E. coli (EPEC) both exploit clathrin mediated endocytosis and the actin cytoskeleton for their disease processes. During L. monocytogenes infections clathrin is used for the microbes to invade their host’s cells and actin filaments are used to generate actin‐rich comet/rocket tails that propel the bacteria within the host cell cytoplasm. When the bacteria hit the host plasma membrane, the comet/rocket tails push the bacteria into neighbouring cells by forming a membrane distention housing the bacteria and the actin tail which forms a corresponding invagination in the neighbouring cell for eventual cell‐to‐cell spreading of the bacteria. EPEC on the other hand remains extracellular and sit atop infected cells on actin‐rich pedestal‐like structures that also recruit clathrin to their apical regions. The FCH and double SH3 domains protein 2 (FCHSD2) functions to activate actin polymerization during clathrin mediated endocytosis. As both L. monocytogenes an EPEC utilize CME and actin polymerization we examined the localization of FCHSD2 during the infections. We found that during L. monocytogenes infections FCHSD2 was absent from the bacterial comet tails, but was prominent at L. monocytogenes actin‐rich membrane protrusions. During EPEC infections FCHSD2 was present within the actin core of the pedestals but did not colocalize with clathrin. Together, this work suggests the FCHSD2 protein has a distinct role at actin‐rich bacterial structures that interact at the membrane which is independent of clathrin.

The actin cytoskeleton is commonly hijacked by bacterial pathogens. Listeria commandeers this filament system during their internalization, and for actin‐rich comet tail formation that enable the microbes to move within the infected cells as well as transfer from cell to cell. Enteropathogenic Escherichia coli (EPEC), an extracellular microbe, controls this cytoskeletal system when they generate E. coli pedestals as part of their firm docking to the plasma membrane of their host's cells and as they “surf” atop the infected cells. Finally, Salmonella exploit the host actin cytoskeleton to enter eukaryotic cells by forcing the generation of actin‐based membrane ruffling. We recently identified Hsc70 in a proteomics screen of EPEC pedestal components. This protein is normally involved with chaperoning functions within the cell. Despite there being a lack of evidence for an association of Hsc70 and actin, due to our proteomics identification of Hsc70 at EPEC pedestals we hypothesized that Hsc70 could be a common target of bacterial pathogens that control the actin cytoskeleton during their disease processes. To test this hypothesis we immunolocalized Hsc70 during Listeria, EPEC and Salmonella infections and found it concentrated at all actin structures generated by the microbes, demonstrating that this protein is a universal component for bacterial actin‐based structures.Support or Funding InformationFunding provided through NSERC. (Grant No. 355316 to J. A. G.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.