Subversion of host cell functions in Listeria

Chromatin modification







[Lebreton et al., Science 2011


The secreted virulence factor LntA targets the nuclear protein BAHD1. LntA localizes to BAHD1-induced heterochromatin foci. BAHD1-YFP and either LntA-V5 or CFP-V5 were cotransfected into C3SV40 cells and detected by immunofluorescence. (D and F) Scale bars, 5 μm.





[Lebreton et al., Science 2011]

 Tandem affinity purification of the BAHD1-associated complex. Solubilized chromatin extracts from HEK293 cells expressing the HPT-BAHD1 fusion or control cells were first purified on anti–protein C affinity matrix, followed by polishing on nickel-Sepharose. Eluted fractions from the first (E1) and second (E2) affinity columns were analyzed by colloidal Coomassie staining (left) or immunoblot (right). I, input; FT, flow-through from first column. Histone H4 was a control for nonspecific binding of chromatin components.







[Ribet et al., Nature 2010]

Decrease in SUMO-conjugated proteins upon Listeria infection

a) Comparison of SUMO-conjugated protein patterns from uninfected HeLa cells (−) or cells infected for 1, 3 or 5 hours with Listeria monocytogenes(L. m.) or Listeria innocua (L. i.). Whole cell lysates were analysed by immunoblotting experiments using antibodies specific for SUMO1 (upper panel) or SUMO2/3 (lower panel) isoforms. b) Analysis of SUMO1-conjugated protein patterns from uninfected HeLa cells (−) or cells infected for 3 hours with wild type (WT), ΔinlB or Δhly L. monocytogenes. c, Analysis of SUMO1 and SUMO2/3-conjugated protein patterns from HeLa cells incubated with 3 nM LLO for 20 min.




[Ribet et al., Cell 2010]

Posttranslational Modification of Host Proteins during Infection Yersinia (blue) is an extracellular pathogen that injects effectors into the host cell’s cytoplasm using a specialized type III secretion system (T3SS). Salmonella (red) triggers its own entry into host cells and replicates in a remodeled vacuole. It also secretes T3SS-dependent effectors. After cell invasion, Listeria (green) escapes from vacuoles and resides free in the cytoplasm, where it replicates and starts moving using the host cell’s actin. Interactions with host factors are mediated by bacterial surface or secreted proteins. Effectors from all three of these bacteria (blue for Yersinia effectors, red for Salmonella effectors, and green for Listeria effectors) alter posttranslational modifications of host proteins (purple) to facilitate pathogens’ replication, propagation, and evasion from host immune responses.





[Ribet et al., Virulence 2010]

Bacterial factors interfering with host cell SUM Oylation. SUM Oylation is a reversible post-translational modification consisting in the covalent linkage of SUM O on a target protein. The conjugation of SUM O requires a set of E1, E2 and E3 SUM O enzymes, whereas deconjugation of SUM O is mediated by SUM O-specific proteases called deSUM Oylases. Some bacterial factors (black diamonds) can interfere with the SUM Oylation of the host cell. The pore-forming toxins LLO, PFO and PLY trigger the degradation of the E2 SUM O enzyme (Ubc9) and the degradation of some host SUM Oylated proteins. XopD and putatively AvrXv4, two factors of X. campestris injected in the host cell cytoplasm during infection, act as deSUM Oylases and lead to the deSUM Oylation of some host proteins.



Histone deacetylation



[Eskandarian et al., Science 2013]

Mechanism and consequence of SIRT2 activation by L. monocytogenes. Listeria induces SIRT2 relocal- ization from cytoplasm to chromatin, where SIRT2 deacetylates H3K18. The consequences of this cascade are control of host transcription, as illustrated by repre- sentative genes regulated by SIRT2, and control of infection, as assessed by staining cells for the secreted bacterial factor InlC (red), which is overexpressed in the cytosol, and host actin, which is polymerized into comet tails by bacteria (green). Error bars indicate SEM; **P < 0.001. Ac, acetyl; deAc, deacetylase.



[Eskandarian et al., Science 2013]

SIRT2 translocates to the nuclear and chromatin fractions upon infection. a) Endog- enous SIRT2 was detected by immunofluorescence in HeLa cells uninfected (-) or infected (+) for 5 hours. Scale bars, 10 mm. DAPI, 4′,6-diamidino-2-phenylindole. b) Uninfected cells (-) or 5-hour infected cells (+) were fractionated and immunoblotted for the indicated proteins. Experiments represent n ≥ 3. c) Immunoprecipitation of SIRT2-FLAG from cytosolic, nuclear, and chromatin fractions was analyzed by MS. The numbers of SIRT2 fragments detected by MS/MS are indicated in the table; numbers of the corresponding SIRT2 peptides are in parentheses. A graphical representation of the MS/MS spectra is also shown.




[Eskandarian et al., Science 2013]

SIRT2 is essential for infection by L. monocytogenes. a)The level of L. monocytogenes infection is measured by FACS analysis of Caco2 cells pretreated (or not) with AGK2, where the geometric mean ( y axis) represents the number of intracellular bacteria (10,000 cells measured; n ≥ 3) at 3, 5, 8, and 24 hours after the start of infection. Error bars denote SEM. *P < 0.05, as measured with a Student’s t test. b) Quantification of immunoblots detecting the bacterial protein InlC in 5-hour–infected HeLa cells knocked down for SIRTs 1, 2, 6, and 7 by siRNA or in cells treated with chemical inhibitors to deacetylases. Error bars indicate SEM. Statistical significance was calculated using a Student’s t test. *P < 0.05; **P < 0.001. c) Caco2 cells were treated (or not) with AGK2 and infected withWT L. monocytogenes for 5 hours. Listeria comet tails are visualized by fluorescence imaging of phalloidin and InlC by immunofluorescence (see text). Images are shown as negatives for better visualization. Scale bars, 50 mm. Large inserted boxes show 5× digital magnifications of the smaller boxes.


Autophagy and Mitochondrial modifications





[Dortet et al., Autophagy 2012]

Strategies used by L. monocytogenes to avoid autophagic recognition. Listeria is able to avoid autophagic recognition using two independent virulence factors, ActA and InlK. Depending on ActA and InlK expression, four possibilities can be distinguished:

(1) ActA and InlK are coexpressed by the bacterium: InlK recruits MVP (red) to the surface of the bacterium. ActA subsequently replaces InlK, and actin 
(green) replaces MVP to disguise the bacteria and prevent ubiquitinated protein (Ub) recruitment/formation, p62 recognition and LC3 recruitment;
(2) neither ActA nor InlK is expressed: Listeria is surrounded by ubiquitinated proteins, p62 and LC3, leading to autophagy;
(3) In the absence of ActA, InlK recruits MVP and efficiently protects bacteria from ubiquitinated protein recruitment/formation, p62 recognition and LC3 recruitment;
(4) ActA is expressed, but not InlK: the recruitment of the Arp2/3 complex and actin is sufficient to prevent ubiquitinated protein recruitment/formation, p62 recognition and LC3 recruitment.





[Stavru et al., PNAS 2011]

Mitochondrial fragmentation upon infection with Listeria monocytogenes.
(A) HeLa cells infected with L. monocytogenes (1 h; MOI of 50) display strongly fragmented mitochondria, whereas cells infected with L. innocua expressing InlB to enter cells do not. Mitochondria were stained with MitoTracker Deep Red (red). L. innocua or L. monocytogenes were stained with polyclonal antibodies R6 and R11 (green). Arrows indicate infected cells, and insets show 2× enlargements. (B) Infection (1 h, MOI of 50) with E.coli(Inv) as a model for Yersinia infection, Shigella flexneri (M09T), Salmonella enterica serovar typhimurium, and enteropathogenic E. coli (EPEC) do not cause mitochondrial fragmentation. Cell nuclei and bacteria were stained with DAPI (blue). Salmonella additionally expresses GFP (green). Mitochondria were stained as in A. Arrows indicate infected cells, and insets show 2× enlargements.

Updated on 13/05/2014



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