Dataset Information

Listeria monocytogenes exploits the MICOS complex subunit Mic10 to promote mitochondrial fragmentation and cellular infection

ABSTRACT: Mitochondrial function adapts to cellular demands and is affected by the ability of the organelle to undergo fusion and fission in response to physiological and non-physiological cues. We previously showed that infection with the human bacterial pathogen Listeria monocytogenes elicits transient mitochondrial fission and a drop in mitochondrial -dependent energy production through a mechanism requiring the bacterial pore-forming toxin listeriolysin O (LLO). Here, we performed quantitative mitochondrial proteomics to search for host factors involved in L. monocytogenes-induced mitochondrial fission. We found that Mic10, a critical component of the mitochondrial contact site and cristae organizing system (MICOS) complex, is significantly enriched in mitochondria isolated from cells infected with wild-type but not with LLO-deficient L. monocytogenes. Increased mitochondrial Mic10 levels did not correlate with upregulated transcription, suggesting a post-transcriptional regulation. We showed that Mic10 is necessary for L. monocytogenes-induced mitochondrial network fragmentation, and that it contributes to L. monocytogenes cellular infection independently of MICOS proteins Mic13, Mic26 and Mic27. Together, L. monocytogenes infection allowed us to uncover a role for Mic10 in mitochondrial fission.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Hct-116 Cell

SUBMITTER: Thibaut Douché

LAB HEAD: Pascale Cossart

PROVIDER: PXD014667 | Pride | 2020-02-17

REPOSITORIES: Pride

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			Action	DRS
	20170720_Q2_TC_ColQ2-28_FC_LLO1.raw	Raw
	20170720_Q2_TC_ColQ2-28_FC_LLO2.raw	Raw
	20170720_Q2_TC_ColQ2-28_FC_LLO3.raw	Raw
	20170720_Q2_TC_ColQ2-28_FC_NI1.raw	Raw
	20170720_Q2_TC_ColQ2-28_FC_NI2.raw	Raw

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Publications

Listeria monocytogenes Exploits Mitochondrial Contact Site and Cristae Organizing System Complex Subunit Mic10 To Promote Mitochondrial Fragmentation and Cellular Infection.

Carvalho Filipe F Spier Anna A Chaze Thibault T Matondo Mariette M Cossart Pascale P Stavru Fabrizia F

mBio 20200204 1

Mitochondrial function adapts to cellular demands and is affected by the ability of the organelle to undergo fusion and fission in response to physiological and nonphysiological cues. We previously showed that infection with the human bacterial pathogen <i>Listeria monocytogenes</i> elicits transient mitochondrial fission and a drop in mitochondrion-dependent energy production through a mechanism requiring the bacterial pore-forming toxin listeriolysin O (LLO). Here, we performed quantitative mi ...[more]

PMID: 32019800

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Project description:Cerebral listeriosis is characterized by neuronal apoptosis and microglial cell activation, but little is known about the bacterial virulence factors involved in this process and how bacterial dissemination is controlled. Here, we show that the cellular target of Listeria monocytogenes (LM) in murine hippocampal cultures is microglia rather than neurons or other glial cells, which are rarely infected. This in vitro model served to demonstrate that infected microglial cells release a soluble factor to the medium responsible for neuronal apoptosis. We investigated the production of this factor in a well-established murine microglia cell model BV2 cells, and compared with J-774 macrophage cells after infection with different LM bacterial mutants. Our purpose was to study in both cell types parameters such as the listericidal capacities, pro-inflammatory cytokines released, and bacterial factors involved in the intracellular cycle. Our data reveal that microglia shows unique features to handle a LM infection. Microglia is three times more permissive for LM intracellular growth than murine macrophages; while producing five times higher levels of pro-inflammatory cytokines IL-6, MCP-1 and TNF-α than macrophages. Using different LM mutants (i.e., LMWT, LM∆LLO, LM∆ActA or LM∆plcB) we conclude that LLO and ActA are not involved in LM proliferation within microglia cells, while they are required for survival within macrophages. Moreover, ActA controls TNF-α production, a cytokine involved in neural apoptosis. Transcriptional differential response of microglia infected with different LM mutants reflected ActA controls the TNF-a signaling pathway through out molecules and chemokines involved in this pathway such as NFkβ, MAPK-1, Ccl2, Ccl3, Ccl4 and CxCl10. The project implies the analysis of two replicates from three different conditions, in total 6 samples: microglia cells non-infected, microglia cells infected with Listeria monocytogenes (LM) wild type (LLO+), microglia cells infected with LM deficient in listeriolysin O (LLO-) and microglia cells infected with LM deficient in ActA (1942). References samples were microglia cells non-infected (NT). Infection with LM was performed for 1 hour, followed by 24 hours in medium with antibiotics to avoid bacterial extracellular growth. Gene expression analysis was performed using Partek Genomics Suite software (version 6.11.0801; Partek). GeneChip data were filtered to remove those probe sets with an intensity raw value close to background levels. Probe sets for each array are pre-processed using RMA. Also, probe sets whose expression change under all experimental conditions was below a threshold, based on the standard desviation of the normalised intensity values, were filtered.

Project description:Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria induce transcriptional changes, in order to modify the set of proteins synthetized by the host cell, or target pre-existing proteins by modulating their post-translational modifications or by triggering their degradation. Protein levels variations in host cells during infection integrates both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the bacterial pathogen Listeria monocytogenes, and more specifically the Listeria toxin Listeriolysin O (LLO). In order to characterize host proteome alterations induced by LLO, we performed a shotgun proteomics analysis on HeLa cells treated or not with the LLO toxin. To this end, we used SILAC labelling (stable isotope labelling by amino acids in cell culture) to compared in a quantitative manner the protein content from two differentially treated cell populations: one control population and one population incubated with a sublytic dose of LLO (3 nM). We decided to expose cells to LLO during only 20 min to limit protein level changes resulting from transcriptional changes. We performed two independent experiments (experiment #1 and experiment #2) with swapped SILAC labelling to rule out putative labelling-dependent effects. A total of 2009 and 1684 proteins was quantified in each independent experiment, respectively, and 1360 proteins were quantified in both experiments. Among these 1360 proteins, we identified a total of 91 proteins for which protein levels were consistently decreased in cells treated with LLO (i.e. with a log2 control/LLO ratio >0.5 in both experiments). To assess whether the host proteasome was involved in the decrease of these 91 proteins, we repeated our proteomics analysis on SILAC-labelled HeLa cells pre-treated with proteasome inhibitors before exposure to LLO. Two independent experiments were similarly performed with swapped SILAC labelling (experiment #3 and experiment #4). Interestingly, we observed that the vast majority of host proteins identified as strongly downregulated in response to LLO also displayed significant decreased levels in the presence of proteasome inhibitors, indicating that the majority of observed decreases in host protein levels induced by LLO are not due to proteasome-mediated degradation. In follow-up experiments, we identified that LLO decreases in particular the protein level of two E2 ubiquitin enzymes, UBE2K and UBE2N, leading to major changes in the host ubiquitylome. This toxin-induced proteome remodeling involves post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. These decreases in host protein levels were observed in different epithelial cell lines but not in macrophages. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data show that different bacterial pore-forming toxins induce deep proteome remodeling, that may impair host epithelial cell functions.

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Project description:Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria induce transcriptional changes, in order to modify the set of proteins synthetized by the host cell, or target pre-existing proteins by modulating their post-translational modifications or by triggering their degradation. Protein levels variations in host cells during infection integrates both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the bacterial pathogen Listeria monocytogenes, and more specifically the Listeria toxin Listeriolysin O (LLO). In order to characterize host proteome alterations induced by LLO, we performed a shotgun proteomics analysis on HeLa cells treated or not with the LLO toxin. To this end, in a first analysis we used SILAC labelling (stable isotope labelling by amino acids in cell culture) to compare in a quantitative manner the protein content from two differentially treated cell populations: one control population and one population incubated with a sublytic dose of LLO (3 nM). We decided to expose cells to LLO during only 20 min to limit protein level changes resulting from transcriptional changes. We performed two independent experiments (experiment #1 and experiment #2) with swapped SILAC labelling to rule out putative labelling-dependent effects. A total of 2,009 and 2,577 proteins was quantified in each independent experiment, respectively, and 1,834 proteins were quantified in both experiments. Among these 1834 proteins, we identified a total of 151 proteins for which protein levels were consistently decreased in cells treated with LLO (i.e. with a log2 control/LLO ratio >0.5 in both experiments). In addition to this preliminary study, we carried out a second experiment using label-free quantitative shotgun proteomics to compare protein abundance in cells treated or not with LLO. Four independent biological replicates were included in this second screen in order to perform a robust statistical analyses of downregulated proteins. Among the 2,973 proteins that were quantified in all independent replicates, we identified a total of 149 proteins (5.0 %) for which protein levels were significantly decreased in cells treated with LLO. In contrast to these decreases, only 16 proteins (0.5 %) showed significant increased levels in LLO-treated cells. This result confirmed that LLO remodels the cell proteome mainly by decreasing the level of host targets. To assess whether the host proteasome was involved in the decrease of these 149 proteins, we repeated our label-free proteomics analysis on HeLa cells pre-treated with proteasome inhibitors before exposure to LLO. Interestingly, we observed that the vast majority (i.e. 83%) of host proteins identified as strongly downregulated in response to LLO also displayed significant decreased levels in the presence of proteasome inhibitors, indicating that the majority of observed decreases in host protein levels induced by LLO are not due to proteasome-mediated degradation. In follow-up experiments, we validated that LLO decreases in particular the protein level of two E2 ubiquitin enzymes, UBE2K and UBE2N, leading to major changes in the host ubiquitylome. This toxin-induced proteome remodeling involves post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. These decreases in host protein levels were observed in different epithelial cell lines but not in macrophages. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data show that different bacterial pore-forming toxins induce deep proteome remodeling, that may impair host epithelial cell functions.