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Stathmin recruits tubulin to Listeria monocytogenes-induced actin comets and promotes bacterial dissemination

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Abstract

The tubulin cytoskeleton is one of the main components of the cytoarchitecture and is involved in several cellular functions. Here, we examine the interplay between Listeria monocytogenes (Lm) and the tubulin cytoskeleton upon cellular infection. We show that non-polymeric tubulin is present throughout Lm actin comet tails and, to a less extent, in actin clouds. Moreover, we demonstrate that stathmin, a regulator of microtubule dynamics, is also found in these Lm-associated actin structures and is required for tubulin recruitment. Depletion of host stathmin results in longer comets containing less F-actin, which may be correlated with higher levels of inactive cofilin in the comet, thus suggesting a defect on local F-actin dynamics. In addition, intracellular bacterial speed is significantly reduced in stathmin-depleted cells, revealing the importance of stathmin/tubulin in intracellular Lm motility. In agreement, the area of infection foci and the total bacterial loads are also significantly reduced in stathmin-depleted cells. Collectively, our results demonstrate that stathmin promotes efficient cellular infection, possibly through tubulin recruitment and control of actin dynamics at Lm-polymerized actin structures.

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Abbreviations

Lm :

Listeria monocytogenes

MT:

Microtubule

ABM:

Actin-based motility

MOI:

Multiplicity of infection

PTM:

Post-translational modifications

STED:

Stimulated emission depletion

ROI:

Region of interest

MFI:

Mean fluorescence intensity

WT:

Wild type

GFP:

Green fluorescent protein

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Acknowledgements

This work received funding from Norte-01-0145-FEDER-000012—Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). ACC and FC were supported by a Fundação para a Ciência e Tecnologia (FCT) Post-doctoral Fellowship (SFRH/BPD/88769/2012) and Ph.D. fellowship (SFRH/BD/61825/2009), respectively, through FCT/MEC co-funded by QREN and POPH (Programa Operacional Potencial Humano). SS was supported by FCT Investigator program (COMPETE, POPH, and FCT). We thank ALM unit from IBMC/i3S for technical support, B. Fonseca (FFUP) for the BeWo cell line and H. Maiato, C. Sunkel and J. B. Relvas laboratories (IBMC/i3S) for sharing reagents. We are also thankful to J. Ferreira and A. Pereira for fruitful discussions.

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  1. Filipe Carvalho

    Present address: Unité des Interactions Bactéries-Cellules, Institut Pasteur, 25 Rue du Dr Roux, 75015, Paris, France

Authors and Affiliations

  1. Group of Molecular Microbiology, i3S-Instituto de Investigação e Inovação em Saúde, IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135, Porto, Portugal

    Ana Catarina Costa, Filipe Carvalho, Didier Cabanes & Sandra Sousa

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  1. Ana Catarina Costa
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ACC, DC and SS conceived and designed the experiments; ACC and FC performed the experiments; ACC, FC, DC and SS analyzed the data; DC and SS obtained the funding; ACC and SS wrote the manuscript.

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Correspondence to Sandra Sousa.

Electronic supplementary material

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18_2018_2977_MOESM1_ESM.tif

Table S1 (relative to Fig. 6b) Stathmin is required for Lm maximal intracellular speed. Control (siC) and stathmin-depleted (siStat) HeLa cells were infected with Lm and imaged by time-lapse microscopy from 7 to 14 h post-infection. Values for Lm speed are mean ± SD of approximately 15 bacteria per condition, in each of the four independent experiments. Fold change (siC/siStat) and statistical significance (p value, Student t test) were calculated (TIFF 156 kb)

18_2018_2977_MOESM2_ESM.tif

Table S2 (relative to Fig. 6d) Stathmin is required for Lm cell-to-cell spreading. Monolayers of control (siC) and stathmin-depleted (siStat) HeLa cells were infected for 24 h with MOI 0.5, fixed and labeled for Lm. Quantification of infection foci areas was performed after multiple image alignment. Values are mean ± SD of at least 60 infection foci areas, per each of the three independent experiments. Fold change (siC/siStat) and statistical significance (p value, Student t test) were calculated (TIFF 114 kb)

18_2018_2977_MOESM3_ESM.tif

Figure S1 (relative to Fig. 1) Tubulin comets are ubiquitously detected in different Lm strains and cell lines. (a to c) Cells were infected for 7 h and subjected to cold shock prior to fixation for immunofluorescence analysis. Representative Z-stack projections of deconvoluted images are shown. Insets shown as high-magnification composite (merge) and single-channel images displaying tubulin (red), actin (green) and DNA (blue). Scale bars represent 10 µm. (a) Caco-2 cells were infected with Lm strains EGD and 10403S. (b) Human (HeLa, Jeg-3, BeWo), murine (J774) and rat kangaroo (Ptk2) cell lines were infected with Lm EGDe. (c) Quantification of the percentage of Lm actin comets and clouds showing tubulin accumulation in HeLa and Ptk2 cells. Results are mean ± SEM of three independent experiments (n ~ 100 per experiment) (TIFF 3262 kb)

18_2018_2977_MOESM4_ESM.tif

Figure S2 (relative to Fig. 2) Nocodazole treatment does not perturb the formation of Lm-associated tubulin comets. Caco-2 cells infected with Lm. Following the bacterial internalization step (1 h), cells were incubated with nocodazole (or DMSO as control) for 6 h, subjected to cold shock, fixed and labeled for tubulin (red), actin (green) and DNA (blue) to be analyzed by fluorescence microscopy. The micrographs are Z axis projections of deconvolutes images. Quantification of the percentage of Lm showing tubulin accumulation together with actin comets is shown in Fig. 2e. Scale bars correspond to 10 µm (TIFF 3340 kb)

18_2018_2977_MOESM5_ESM.tif

Figure S3 (relative to Fig. 4) Efficiency of stathmin depletion in HeLa cells. (a) Western blot analysis of stathmin expression in HeLa cells treated with control (siC) or with stathmin (siStat) siRNA. GAPDH levels were used as loading control. (b) Quantification of siRNA-mediated silencing of stathmin as evaluated by densitometric analysis of Western blot bands, normalized for GAPDH expression. Results are mean ± SD from four independent experiments. Asterisks indicate statistical significance as determined by Student t test: p < 0.0001 (****) (TIFF 95 kb)

18_2018_2977_MOESM6_ESM.tif

Figure S4 (relative to Fig. 5) Efficiency of stathmin depletion in HeLa cells. Stathmin expression levels evaluated by densitometric analysis of Western blot bands, normalized for GAPDH expression. Results are mean ± SD of four independent experiments. Asterisks indicate statistical significance as determined by Student t test: p < 0.01 (**) (TIFF 50 kb)

18_2018_2977_MOESM7_ESM.avi

Movie S1 (relative to Fig. 3) Tubulin coats actin comet tails polymerized by Lm. HeLa cells infected with Lm were labeled for tubulin (red), Lm (WGA, cyan) and actin (SiRactin, green). Movie shows a 360º rotation of the Lm comet tail obtained by three-dimensional reconstitution of stimulated emission depletion (STED) super-resolution microscopy images (AVI 138248 kb)

18_2018_2977_MOESM8_ESM.avi

Movies S2 and S3 (relative to Fig. 6a, b and Table S1) Stathmin controls Lm speed. Control (Movie S2) and stathmin-depleted cells (Movie S3) infected with Lm were subjected to phase-contrast live-imaging from 7 to 14 h post-infection. Arrows indicate the position of Lm throughout time and the black line shows the described path. Videos are reproduced at 5 frames per second (AVI) (AVI 181 kb)

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Costa, A.C., Carvalho, F., Cabanes, D. et al. Stathmin recruits tubulin to Listeria monocytogenes-induced actin comets and promotes bacterial dissemination. Cell. Mol. Life Sci. 76, 961–975 (2019). https://doi.org/10.1007/s00018-018-2977-7

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