The intracellular bacterium Listeria monocytogenes hijacks the actin polymerization machinery within human cells for its entry, intracellular movement and subsequent spreading from cell‐to‐cell. Intracellular motility of Listeria involves Arp2/3‐mediated actin polymerization at one pole of the bacteria, resulting in the generation of structures known as comet tails. During cell‐to‐cell spreading, comet tails protrude from the host cell membrane from structures referred to as listeriopods. Although the minimal protein constituents required for these actin‐based structures were identified years ago, a complete understanding of the molecular constituents and their specific functions at these sites remains to be investigated. Cyclophilin A (CypA) is a cytosolic protein involved in protein folding and potentially actin nucleation. Thus, because Listeria depends on actin‐based structures for crucial events in its disease process, we tested the hypothesis that CypA is recruited to Listeria comet tails and sites of cell‐to‐cell bacterial spreading. Through the use of fluorescently‐tagged CypA DNA constructs and time‐lapse imaging, we demonstrate that CypA is present at 1) the surface of Listeria prior to comet tail formation, 2) comet tails themselves and 3) listeriopods. Our data reveals a new component in the actin‐based motility of Listeria and suggests that CypA may play a crucial role in the cell‐to‐cell spreading of Listeria.Grant Funding Source: NSERC and departmental funds
Listeria monocytogenes (Listeria) co‐opts the actin cytoskeleton of host cells for their entry, intracellular motility and dissemination. These bacteria generate Arp2/3‐based, branched actin‐containing structures called comet tails that propel the bacteria within and amongst host cells. The host protein palladin is a crucial component of actin‐rich structures. Whether this protein can promote actin based motility events remains unclear. Here we tested the hypothesis that palladin is a crucial organizer of actin structures generated during Listeria infections and simultaneously used Listeria as a model to establish if palladin could compensate for processes requiring the Arp2/3 complex. Using immunofluorescence microscopy we identified palladin at bacterial invasion sites and motile comet tails. Strikingly, depletion of palladin via RNAi results in shorter and severely misshapen comet tails. When palladin mutants defective for actin or VASP binding are expressed in cells, comet tails disintegrate or become progressively thinner as they move. Through transmission electron microscopy, we demonstrate that these thin comet tails result from a switch in the actin network from branched arrays to parallel F‐actin bundles. To determine whether palladin could compensate for the Arp2/3 complex during comet tail motility, we overexpressed palladin in cells treated with the potent Arp2/3 inhibitor CK‐666. In these cells, Listeria can initiate and maintain its actin‐based motility. Additionally, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells Listeria fail to generate comet tails. However, when palladin is overexpressed in this Arp2/3 functionally null cell line, the ability of Listeria to generate actin clouds and comet tails is restored. Taken together, we show that palladin is essential for the organization of F‐actin structures generated by Listeria and, importantly, can compensate for Arp2/3 complex functional defects without hindrance during bacterial actin‐based motility.Support or Funding InformationGrant Funding Source: NSERC (grant no. 355316 to J.A.G and grant no. 155397 to A.W.V), NIH (grant no. R15 GM120670 to M.R.B) and, SFU departmental fundsThis 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 (Listeria) bacteria kill 25% of people they infect. To cause disease, these microbes enter their host's cells and commandeer various components of those cells. Importantly, the bacteria generate motile actin‐rich structures attached to their own surface in order to protrude from the host cell's plasma membrane as they attempt to spread into neighboring cells for further infections. These actively spreading bacteria reside within the tips of protrusive actin‐rich structures called listeriopods. To better understand infections caused by Listeria, current research is targeted towards unravelling the precise molecular components involved in the formation of listeriopods. In a screen of proteins found at actin‐rich structures formed by pathogenic Escherichia coli (E. coli), we identified the eukaryotic peptidyl prolyl cis‐trans isomerase (PPIase), cyclophilin A (CypA). Here we tested the hypothesis that CypA is a crucial protein needed to propagate infections by Listeria. Using CypA antibodies we demonstrate that this protein is concentrated specifically at listeriopods. To test the functional importance of CypA at these structures we infected CypA null murine fibroblasts with Listeria and saw that listeriopods were structurally collapsed and twisted. Ultrastructural examination revealed wide‐scale alterations to the organization of actin filaments within the core of the collapsed listeriopods. Importantly, when CypA is re‐expressed in these null cells, listeriopods appear morphologically normal. We used a Listeria cell‐to‐cell spreading assay to examine the ability of listeriopods to protrude into neighboring uninfected cells and found a ~2.5‐fold decrease in the proportion of listeriopods moving cell‐to‐cell when they were generated in CypA knock‐out cells. In conclusion, we have shown that CypA is a key protein needed for bacterial cell‐to‐cell dissemination.Support or Funding InformationGrant Funding Source: NSERC (grant no. 355316 to J.A.G), SFU departmental funds and SFU Multi‐Year Funding (A.S.D)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Listeria monocytogenes (Listeria) bacteria are well‐known for their ability to hijack the eukaryotic actin cytoskeleton during infections. To propagate disease, Listeria generate Arp2/3‐based actin‐rich structures called comet tails that move the bacteria within and amongst host cells. The host protein palladin is a key component of actin‐rich structures normally generated during eukaryotic cell motility, however the precise function(s) of palladin during motility remains unclear. Here we tested the hypothesis that palladin is a crucial factor for Listeria motility and simultaneously used Listeria as a model to determine if palladin itself could functionally replace the Arp2/3 complex. Using palladin‐targeting antibodies, we identified palladin at Listeria invasion sites and comet tails. Strikingly, when we depleted cells of palladin, comet tails became shorter and severely misshapen. In cells expressing palladin mutants defective for actin or VASP binding, comet tails began to disintegrate or became progressively thinner as they moved. Ultrastructural examination of these thin comet tails revealed a switch in the comet tail actin network from highly branched arrays to parallel bundles. To test whether palladin could compensate for the Arp2/3 complex during bacterial motility, we overexpressed palladin in cells treated with the potent Arp2/3 inhibitor CK‐666. In these cells Listeria motility was unperturbed. Next we used a cell line depleted of several Arp2/3 complex subunits. As expected, Listeria were non‐motile during infections of these cells, however palladin overexpression in this Arp2/3 functionally null cell line restored the ability of Listeria to generate the actin‐rich structures formed by the bacteria. To definitively demonstrate palladin's ability to compensate for a lack of functional Arp2/3, we used purified protein components in conjunction with Listeria bacteria to show that actin‐rich structures formed by Listeria are generated in a cell‐free system containing palladin in place of the Arp2/3 complex. In conclusion, we show that palladin structurally organizes bacterial actin‐rich structures and importantly, compensates for the Arp2/3 complex without hindrance during bacterial actin‐based motility.Support or Funding InformationGrant Funding Source: NSERC (grant no. 355316 to J.A.G and grant no. 155397 to A.W.V), NIH (grant no. R15 GM120670 to M.R.B), SFU departmental funds and SFU Multi‐Year Funding (A.S.D)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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