Rapid Remodeling of the Host Epithelial Cell Proteome by the Listeriolysin O (LLO) Pore-forming Toxin.

Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria modulate host gene transcription, thereby modifying the set of proteins synthetized by the infected cell. Bacteria can also target pre-existing host proteins and modulate their post-translational modifications or trigger their degradation. Analysis of protein levels variations in host cells during infection allows to integrate both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the toxin Listeriolysin O (LLO), secreted by the bacterial pathogen Listeria monocytogenes. We showed that a short-term treatment with LLO remodels the host cell proteome by specifically decreasing the abundance of 149 proteins. The same decrease in host protein levels was observed in different epithelial cell lines but not in macrophages. We show in particular that this proteome remodeling affects several ubiquitin and ubiquitin-like ligases and that LLO leads to major changes in the host ubiquitylome. Strikingly, this toxin-induced proteome remodeling involves only post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. 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 reveal that different bacterial pore-forming toxins induce important host proteome remodeling, that may impair epithelial cell functions.

p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways.

Autophagy is an important mechanism of innate immune defense. We have recently shown that autophagy components are recruited with septins, a new and increasingly characterized cytoskeleton component, to intracytosolic Shigella that have started to polymerize actin. On the other hand, intracytosolic Listeria avoids autophagy recognition by expressing ActA, a bacterial effector required for actin polymerization. Here, we exploit Shigella and Listeria as intracytosolic tools to characterize different pathways of selective autophagy. We show that the ubiquitin-binding adaptor proteins p62 and NDP52 target Shigella to an autophagy pathway dependent upon septin and actin. In contrast, p62 or NDP52 targets the Listeria ActA mutant to an autophagy pathway independent of septin or actin. TNF-α, a host cytokine produced upon bacterial infection, stimulates p62-mediated autophagic activity and restricts the survival of Shigella and the Listeria ActA mutant. These data provide a new molecular framework to understand the emerging complexity of autophagy and its ability to achieve specific clearance of intracytosolic bacteria.

K+ efflux is required for histone H3 dephosphorylation by Listeria monocytogenes listeriolysin O and other pore-forming toxins.

Chromatin modification triggered by bacteria is a newly described mechanism by which pathogens impact host transcription. Listeria monocytogenes dephosphorylates histone H3 through the action of listeriolysin O (LLO); however, the underlying mechanism is unknown. Here we show that an unrelated pore-forming toxin, Aeromonas aerolysin, also provokes H3 dephosphorylation (dePH3). As reported for aerolysin, we show that LLO and related toxins induce a pore-dependent K(+) efflux and that this efflux is the signal required for dePH3. In addition, LLO-induced K(+) efflux activates caspase-1. However, we demonstrate that dePH3 is unlinked to this activation. Therefore, our study unveils K(+) efflux as an important signal leading to two independent events critical for infection, inflammasome activation and histone modification.

Single-cell techniques using chromosomally tagged fluorescent bacteria to study Listeria monocytogenes infection processes.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen which invades different cell types, including nonphagocytic cells, where it is able to replicate and survive. The different steps of the cellular infectious process have been well described and consist of bacterial entry, lysis of the endocytic vacuole, intracellular replication, and spreading to neighboring cells. To study the listerial infectious process, gentamicin survival assays, plaque formation, and direct microscopy observations are typically used; however, there are some caveats with each of these techniques. In this study we describe new single-cell techniques based on use of an array of integrative fluorescent plasmids (green, cyan, and yellow fluorescent proteins) to easily, rapidly, and quantitatively detect L. monocytogenes in vitro and in vivo. We describe construction of 13 integrative and multicopy plasmids which can be used for detecting intracellular bacteria, for measuring invasion, cell-to-cell spreading, and intracellular replication, for monitoring in vivo infections, and for generating transcriptional or translational reporters. Furthermore, we tested these plasmids in a variety of epifluorescence- and flow cytometry-based assays. We showed that we could (i) determine the expression of a particular promoter during the cell cycle, (ii) establish in one rapid experiment at which step in the cell cycle a particular mutant is defective, and (iii) easily measure the number of infected cells in vitro and in mouse organs. The plasmids that are described and the methods to detect them are new powerful tools to study host-Listeria interactions in a fast, robust, and high-throughput manner.

Revealing eukaryotic histone-modifying mechanisms through bacterial infection.

In the long co-evolution of host-pathogen interaction, bacteria have developed sophisticated strategies to manipulate host cell mechanisms and reprogram host transcription. Targeting chromatin, mainly through post-translational modification (PTM) of histone proteins, is one strategy that has been revealed over the last decade. Indeed, histone modifications play a crucial role in regulating transcription during cell type and stimulus specific responses, making them good targets during infection. Therefore, the study of host-pathogen interactions provides breakthroughs in understanding virulence mechanisms, but also in host cell mechanisms. Although chromatin is regulated by DNA methylation, noncoding RNAs, and post-translational modifications of histones, most studies have concentrated on bacteria-induced histone modifications, which will be the focus of this review. We will discuss the different mechanisms used by bacteria to induce histone PTMs, whether it is through direct targeting of pathogen effector enzymes, or indirectly through modulation of cellular signaling cascade. We will summarize the concepts we learned in cell biology from exploring bacteria-triggered histone modifications, by focusing on the signaling cascades modified by bacteria, bacterial mimics of eukaryotic enzymes, and the novel histone marks imposed upon infection.

Streptococcus pneumoniae Infection Promotes Histone H3 Dephosphorylation by Modulating Host PP1 Phosphatase.

Pathogenic bacteria can alter host gene expression through post-translational modifications of histones. We show that a natural colonizer, Streptococcus pneumoniae, induces specific histone modifications, including robust dephosphorylation of histone H3 on serine 10 (H3S10), during infection of respiratory epithelial cells. The bacterial pore-forming toxin pneumolysin (PLY), along with the pyruvate oxidase SpxB responsible for H2O2 production, play important roles in the induction of this modification. The combined effects of PLY and H2O2 trigger host signaling that culminates in H3S10 dephosphorylation, which is mediated by the host cell phosphatase PP1. Strikingly, S. pneumoniae infection induces dephosphorylation and subsequent activation of PP1 catalytic activity. Colonization of PP1 catalytically deficient cells results in impaired intracellular S. pneumoniae survival and infection. Interestingly, PP1 activation and H3S10 dephosphorylation are not restricted to S. pneumoniae and appear to be general epigenomic mechanisms favoring intracellular survival of pathogenic bacteria.

H3K4me1 Supports Memory-like NK Cells Induced by Systemic Inflammation.

Natural killer (NK) cells are unique players in innate immunity and, as such, an attractive target for immunotherapy. NK cells display immune memory properties in certain models, but the long-term status of NK cells following systemic inflammation is unknown. Here we show that following LPS-induced endotoxemia in mice, NK cells acquire cell-intrinsic memory-like properties, showing increased production of IFNγ upon specific secondary stimulation. The NK cell memory response is detectable for at least 9 weeks and contributes to protection from E. coli infection upon adoptive transfer. Importantly, we reveal a mechanism essential for NK cell memory, whereby an H3K4me1-marked latent enhancer is uncovered at the ifng locus. Chemical inhibition of histone methyltransferase activity erases the enhancer and abolishes NK cell memory. Thus, NK cell memory develops after endotoxemia in a histone methylation-dependent manner, ensuring a heightened response to secondary stimulation.

Listeriolysin O: the Swiss army knife of Listeria.

Listeriolysin O (LLO) is a toxin produced by Listeria monocytogenes, an opportunistic bacterial pathogen responsible for the disease listeriosis. This disease starts with the ingestion of contaminated foods and mainly affects immunocompromised individuals, newborns, and pregnant women. In the laboratory, L. monocytogenes is used as a model organism to study processes such as cell invasion, intracellular survival, and cell-to-cell spreading, as this Gram-positive bacterium has evolved elaborate molecular strategies to subvert host cell functions. LLO is a major virulence factor originally shown to be crucial for bacterial escape from the internalization vacuole after entry into cells. However, recent studies are revisiting the role of LLO during infection and are revealing new insights into the action of LLO, in particular before bacterial entry. These latest findings along with their impact on the infectious process will be discussed.

Entrapment of intracytosolic bacteria by septin cage-like structures.

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.

Histone modifications and chromatin remodeling during bacterial infections.

The link between bacteria and host chromatin remodeling is an emerging topic. The exciting recent discoveries on bacterial impact on host epigenetics, as discussed in this Review, highlight yet another strategy used by bacterial pathogens to interfere with key cellular processes. The study of how pathogens provoke host chromatin changes will also provide new insights into host epigenetic regulation mechanisms.

Histone modifications induced by a family of bacterial toxins.

Upon infection, pathogens reprogram host gene expression. In eukaryotic cells, genetic reprogramming is induced by the concerted activation/repression of transcription factors and various histone modifications that control DNA accessibility in chromatin. We report here that the bacterial pathogen Listeria monocytogenes induces a dramatic dephosphorylation of histone H3 as well as a deacetylation of histone H4 during early phases of infection. This effect is mediated by the major listerial toxin listeriolysin O in a pore-forming-independent manner. Strikingly, a similar effect also is observed with other toxins of the same family, such as Clostridium perfringens perfringolysin and Streptococcus pneumoniae pneumolysin. The decreased levels of histone modifications correlate with a reduced transcriptional activity of a subset of host genes, including key immunity genes. Thus, control of epigenetic regulation emerges here as an unsuspected function shared by several bacterial toxins, highlighting a common strategy used by intracellular and extracellular pathogens to modulate the host response early during infection.