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Listeria monocytogenes hijacks CD147 to ensure proper membrane protrusion formation and efficient bacterial dissemination

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Abstract

Efficient cell-to-cell transfer of Listeria monocytogenes (L. monocytogenes) requires the proper formation of actin-rich membrane protrusions. To date, only the host proteins ezrin, the binding partner of ezrin, CD44, as well as cyclophilin A (CypA) have been identified as crucial components for L. monocytogenes membrane protrusion stabilization and, thus, efficient cell-to-cell movement of the microbes. Here, we examine the classical binding partner of CypA, CD147, and find that this membrane protein is also hijacked by the bacteria for their cellular dissemination. CD147 is enriched at the plasma membrane surrounding the membrane protrusions as well as the resulting invaginations generated in neighboring cells. In cells depleted of CD147, these actin-rich structures appear similar to those generated in CypA depleted cells as they are significantly shorter and more contorted as compared to their straighter counterparts formed in wild-type control cells. The presence of malformed membrane protrusions hampers the ability of L. monocytogenes to efficiently disseminate from CD147-depleted cells. Our findings uncover another important host protein needed for L. monocytogenes membrane protrusion formation and efficient microbial dissemination.

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Acknowledgements

We thank Pascale Cossart for providing L. monocytogenes strains (EGD BUG 600 wild-type and ΔactA mutant), Calvin Roskelley for providing the SKOV3 cells, and Tatiana Pushkarsky for preparing the CD147 plasmids.

Funding

This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada (355316 and RGPIN-2018-05100 to JAG), as well as SFU Departmental Funds to JAG. ASD is an SFU Multi-year funding award recipient.

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Authors and Affiliations

  1. Department of Biological Sciences, Centre for Cell Biology, Development, and Disease, Simon Fraser University, Burnaby, BC, Canada

    Aaron S. Dhanda, Katarina T. Lulic, Connie Yu & Julian A. Guttman

  2. Dental and Craniofacial Research Institute and School of Dentistry, University of California, Los Angeles, Los Angeles, CA, USA

    Robert H. Chiu

  3. Surgical Oncology and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA, USA

    Robert H. Chiu

  4. The George Washington University School of Medicine and Health Sciences, 2300 Eye St NW, Washington, DC, 20037, USA

    Michael Bukrinsky

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  1. Aaron S. Dhanda
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  2. Katarina T. Lulic
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  3. Connie Yu
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  4. Robert H. Chiu
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  5. Michael Bukrinsky
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  6. Julian A. Guttman
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Contributions

ASD and JAG conceived the study. KTL provided results for Figs. S6a–c and CY provided results for Fig. S4b. ASD performed all other experiments. RHC and MB provided CypA and CD147 reagents as well as expertise. All authors analyzed the data and wrote the manuscript.

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Correspondence to Julian A. Guttman.

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18_2019_3130_MOESM1_ESM.jpg

Supplemental Fig. 1 Primary antibody and GFP vector controls do not localize to L. monocytogenes membrane protrusions or invaginations. (A) HeLa cells were infected with wild-type L. monocytogenes for 6 h. Samples were fixed then stained with normal mouse IgG antibody (green), Alexa594-phalloidin (red) to visualize F-actin, and DAPI (blue) to visualize DNA. The control IgG is not enriched at the plasma membrane surrounding membrane protrusions (top) or at invaginations (bottom). Open arrowheads indicate L. monocytogenes bacteria (blue), while solid arrowheads point to protruding bacteria (red). Scale bar is 5 μm. (B) HeLa cells transfected with GFP alone (green) were infected with wild-type L. monocytogenes for 6 h. They were then fixed and stained with Alexa594-phalloidin (red) to visualize F-actin and DAPI (blue) to visualize DNA. GFP alone is not enriched at the plasma membrane surrounding the membrane protrusions (solid arrowhead) or at invaginations (open arrowhead). Scale bar is 5 μm. (C) HeLa cells transfected with pmKate2-f-mem alone (red) were infected with wild-type L. monocytogenes for 6 h. Signal from pmKate2-f-mem at L. monocytogenes membrane protrusions (bottom solid arrowhead) is visibly decreased compared to pmKate2-f-mem at normal host membrane structures (membrane ruffles, orange arrowheads; filopodia, yellow arrowheads). Open arrowhead indicates location of L. monocytogenes bacterium. Scale bar is 5 μm

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Supplemental Fig. 2 CD147 is enriched at sites of L. monocytogenes cell-to-cell spreading in Caco-2 cells. Caco-2 cells were infected with wild-type L. monocytogenes for 5 h and then fixed and stained with mouse monoclonal CD147-targeting antibodies (green), Alexa594-phalloidin (red) to visualize F-actin, and DAPI (blue) to visualize DNA. CD147 was enriched at the plasma membrane surrounding membrane protrusions. The open arrowhead indicates the L. monocytogenes bacterium (blue). The solid arrowhead points to CD147 at the membrane protrusion. Scale bar is 5 μm

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Supplemental Fig. 3 CD147 is not recruited by L. monocytogenes bacteria that are unable to induce F-actin polymerization. HeLa cells were infected with ΔactA L. monocytogenes for 6 h, and fixed and then stained with mouse monoclonal CD147 targeting antibodies (green), DAPI (blue) to visualize bacteria, and Alexa594-phalloidin (red) to visualize F-actin. CD147 is absent from the surface of intracellular bacteria (solid arrowhead). Scale bar is 10 μm

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Supplemental Fig. 4 In cells depleted of CD147 comet/rocket tails remain morphologically normal. (A) HeLa cells were treated with non-targeting [control] (Ctrl siRNA) or CD147-targeted (CD147 siRNA) siRNA duplexes, and whole-cell lysates were collected and probed for endogenous CD147 using mouse monoclonal CD147-targeting antibodies. Loading control with α-tubulin is shown below. (B) Quantification of CD147 protein levels from HeLa cells treated with non-targeting [control] (Ctrl siRNA) or CD147-targeted (CD147 siRNA) siRNA duplexes. Duplicate lanes of lysate (from three independent blots) were analyzed. Percent values (relative to control, [± s.d.]) are 100% [Ctrl siRNA] and 10% [CD147 siRNA] **, P < 0.01 (unpaired Mann–Whitney U test). (C) Cytosolic comet/rocket tails (solid arrowheads) appear morphologically normal in cells treated with CD147-targeting siRNA sequences. Open arrowheads indicate contorted L. monocytogenes membrane protrusions. Scale bar is 10 μm

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Supplemental Fig. 5 L. monocytogenes membrane protrusions are contorted in SKOV3 and A549 cell lines depleted of CD147. SKOV3 (A) and A549 (B) cells were treated with non-targeting [Ctrl siRNA] or CD147 targeting [CD147 siRNA] siRNA sequences, infected with wild-type L. monocytogenes for 6 h, fixed, and then stained with mouse monoclonal CD147 targeting antibodies (green), DAPI (blue) to visualize bacteria (open arrowheads), and Alexa594-phalloidin (red) to visualize F-actin and membrane protrusions. CD147 is present at the plasma membrane of normal protrusions (solid arrowheads) extending from [Ctrl siRNA]-treated cells. Membrane protrusions (solid arrowheads) are contorted in CD147-depleted [CD147 siRNA] cells (B). Scale bars are 5 μm

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Supplemental Fig. 6 Depletion of CD147 does not affect ezrin or cyclophilin A recruitment to membrane protrusions. (A) Ezrin immunolocalization (green) at actin-rich L. monocytogenes membrane protrusions (red) generated in cells treated with non-targeting control [Ctrl siRNA] or CD147-targeting [CD147 siRNA] siRNA sequences. Ezrin is present within the core of normal as well as misshaped L. monocytogenes membrane protrusions. Open arrowheads indicate the location of L. monocytogenes bacteria (blue) within membrane protrusions of interest. Scale bar is 10 μm. (B) Enlargement of boxed regions from (A). Open arrowheads indicate the location of L. monocytogenes bacteria (blue), while solid arrowheads point to the membrane protrusions. Scale bar is 5 μm. (C) Enlargement of L. monocytogenes membrane protrusions from numbered arrows in (B). Solid arrowheads indicate morphologically altered membrane protrusions (red). Color intensities were enhanced from (B) to clearly identify the bacteria and membrane protrusions. Scale bar is 2 μm

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Supplemental Fig. 7 CD147 and Cyclophilin A Immunolocalization within L. monocytogenes membrane protrusions. L. monocytogenes bacteria at varying stages of membrane protrusion formation highlighting the recruitment patterns of CypA (red) and CD147 (green). Shown in box 1, bacteria (white open arrow) initially contacting the host plasma membrane show CD147 staining as bright clustering at the bacterial pole in contact with the cell surface (green arrows) and CypA at the opposite pole where the actin comet/rocket tail originates (red arrow). As the bacteria begin to protrude from the cell surface (box 2), recruitment continues with CD147 at the membrane-facing and CypA at the cytoplasmic-facing pole of the bacteria. CypA is enriched throughout the actin tail that eventually pushes the bacteria past the host cell edge; CD147 remains solely at the membrane surrounding the protrusion (box 3). CypA and CD147 appear to occupy similar regions specifically at zones where the membrane is in close proximity to the comet/rocket tail–bacteria interface (box 3, arrowhead). As membrane protrusions begin to extend further, CypA localization remains within the actin-rich core, while CD147 continues to be recruited around the entirety of the protrusion membrane. This localization continues until the point where the membrane protrusion extends far from the cell body (boxes 4–6). Zones where the actin narrows together with areas where the membrane appear to merge together show intermixing of CypA and CD147 staining. Scale bar is 1 μm

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Supplemental Fig. 8 Antibody blocking of CD147 results in the appearance of collapsed membrane protrusions. (A) HeLa cells infected with wild-type L. monocytogenes for 2 h, washed and then incubated with media-containing either the CD147-targeting antibody [MEM-M6/1; 5 µg/mL] or normal mouse IgG antibody [5 µg/mL] for an additional 5 h. Samples were fixed then stained with the mouse monoclonal CD147-targeting antibody (green), Alexa594-phalloidin (red) to visualize F-actin and DAPI (blue) to visualize DNA. L. monocytogenes membrane protrusions are collapsed when cells are treated with the CD147 blocking antibody (top). CD147 maintains its localization at membrane protrusions (solid arrowheads). Open arrowheads indicate bacteria within membrane protrusions. Scale bar is 2 μm. (B) HeLa cells infected with wild-type L. monocytogenes for 2 h, washed, and then incubated with media-containing either the CD147-targeting antibody [MEM-M6/1; 5 µg/mL] or normal mouse IgG antibody [5 µg/mL] for an additional 5 h. Samples were fixed and then stained with the rabbit polyclonal CypA-targeting antibody (green), Alexa594-phalloidin (red) to visualize F-actin, and DAPI (blue) to visualize DNA. CypA maintains its localization at collapsed as well as at morphologically normal membrane protrusions (solid arrowheads). Open arrowheads indicate bacteria within membrane protrusions. Scale bar is 2 μm

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Supplemental Fig. 9 The CypA-binding site of CD147 is dispensable for L. monocytogenes membrane protrusion formation and CypA recruitment to the structures. (A) Mixed cells assays where Hela cells infected with wild-type L. monocytogenes and expressing the GFP-CD147-P211A plasmid (CypA-binding deficient) were overlaid onto naïve, uninfected wild-type HeLa cells (top) or reverse experiments where untransfected infected HeLa cells were overlaid onto GFP-CD147-P211A expressing uninfected HeLa cells (bottom). GFP-CD147-P211A localizes to morphologically normal membrane protrusions (solid arrowhead; membrane protrusion sending cell [bacterium indicated by open arrowhead]) as well as protrusion invaginations (open arrowheads; membrane protrusion receiving cell). Scale bar is 5 μm. (B) HeLa cells infected with wild-type L. monocytogenes and expressing the GFP-CD147-P211A plasmid (green) were fixed and stained with a rabbit polyclonal CypA-targeting antibody (blue) and Alexa350-phalloidin (red) to visualize F-actin. CypA maintains its localization at membrane protrusions (solid arrowhead) generated in these cells. Scale bar is 2 μm. (C) The presence of CypA (top) or CD147 (bottom) at malformed L. monocytogenes membrane protrusions is not perturbed by siRNA depletion of CD147 (CD147 siRNA) or genetic deletion of CypA (CypA KO), respectively. Open arrowheads indicate the location of L. monocytogenes bacteria (blue). Scale bar is 2 μm. (D) Quantification of results from (C). Percent frequency of protein localization is 94% (left) and 99% (right). The number of membrane protrusions analyzed is as follows: 97 (from 15 CD147 siRNA-treated cells) and 79 (from 15 CypA KO cells)

18_2019_3130_MOESM10_ESM.jpg

Supplemental Fig. 10 α-Actinin 4 is recruited to the actin-rich core of L. monocytogenes membrane protrusions as well to actin clouds and comet/rocket tails. (A) HeLa cells infected with wild-type L. monocytogenes for 6 h (to observe membrane protrusion formation) were fixed then stained with a rabbit monoclonal α-actinin 4 targeting antibody (green), Alexa594-phalloidin (red) to visualize F-actin, and DAPI (blue) to visualize DNA. CD147 is enriched within the core of the membrane protrusions. The solid and open arrowheads indicate the membrane protrusion of interest and its associated bacterium, respectively. Scale bar is 5 μm. (B) Hela cells infected and stained exactly as in (A). α-actinin 4 is enriched at actin clouds surrounding stationary bacteria (open arrowhead) and comet/rocket tails of motile cytosolic bacteria (solid arrowhead). Insets depict enlargement of boxed regions. Scale bars are 10 μm and 2 μm (inset)

18_2019_3130_MOESM11_ESM.jpg

Supplemental Fig. 11 Confirmation of CD147 depletion in cells during L. monocytogenes cell-to-cell spreading experiments. The same images displayed as in Fig. 3A but showing that CD147 is highly depleted in L. monocytogenes infected cells treated with CD147-targeting (CD147 siRNA) siRNA sequences compared to infected cells treated with non-targeting control siRNA sequences (Ctrl siRNA). Scale bar is 20 μm

18_2019_3130_MOESM12_ESM.jpg

Supplemental Fig. 12 CD147 is recruited to the plasma membrane of S. flexneri actin-rich membrane protrusions and is crucial for efficient cell-to-cell spreading of the bacteria. (A) HeLa cells infected with wild-type S. flexneri for 2.5 h were fixed and then stained with a mouse monoclonal CD147 targeting antibody (green), Alexa594-phalloidin (red) to visualize F-actin and DAPI (blue) to visualize DNA. CD147 is enriched at the plasma membrane surrounding membrane protrusions. Scale bar is 5 μm. (B) Quantification of S. flexneri intercellular spreading assays. The number of infected cells contained within foci from HeLa cells treated with non-targeting [control] (Ctrl siRNA) or CD147-targeting (CD147 siRNA) siRNA duplexes. 26 and 28 foci from cells treated with non-targeting [control] and CD147-targeting siRNA, respectively, were imaged and analyzed. Average percent values (relative to control, [± s.d.]) are 100% [Ctrl siRNA] and 76.5% [CD147 siRNA]. ***, P < 0.0001 (unpaired Mann–Whitney U test)

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Dhanda, A.S., Lulic, K.T., Yu, C. et al. Listeria monocytogenes hijacks CD147 to ensure proper membrane protrusion formation and efficient bacterial dissemination. Cell. Mol. Life Sci. 76, 4165–4178 (2019). https://doi.org/10.1007/s00018-019-03130-4

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