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Bacterial, cellular and epigenetic factors that control enteropathogenicity

Periodic Reporting for period 3 - BacCellEpi (Bacterial, cellular and epigenetic factors that control enteropathogenicity)

Reporting period: 2018-10-01 to 2019-09-30

- Understanding the establishment and persistence of bacterial infections in particular food borne infections which start in the gut, requires integrating an ensemble of factors including bacterial factors and host components and elucidating what are the other microorganisms present in the intestinal tract, i.e. the microbiota. We have focused our analysis on the infection by a gut pathogen Listeria monocytogenes that we used as a model system since many years.
- Such a research programme is important because antibiotic resistance is increasing and raises critical public health problems in particular for several intestinal infections.
- In this project, we intended to analyze 1) the specific attributes of the bacterium and in particular its specific proteins at work during infection compared to life in the environment ; 2) the behavior of intracellular organelles critical for survival of the host cell, in particular mitochondria ; 3) the modification of the host genome expression induced by histone modifications, modifications of the transcriptome and whether there is a cellular memory of infection.

Our project has led to important findings : we have discovered a never reported bacterial mini protein and showed that this mini protein can relay an extracellular stress to the intracellular nanomachine called the stressosome and coordinate expression of a series of genes involved in the stress response. In addition, we have determined the atomic structure of the Listeria stressosome, the largest bacterial nanomachine after the ribosome. We have discovered and characterized the first secreted bacterial RNA-binding protein which during infection binds and activates RIG-I and leads to the production of type I interferon. We have identified in the Listeria strains responsible for epidemics, a bacteriocin which acts in the gut and favors infection. We then discovered a second bacteriocin which modulates infection by targeting Prevotella an abundant gut commensal. We have discovered a novel mechanism of antibiotic resistance , i.e. ribosome splitting.
We have also discovered new host components playing a key role in infection. Mitochondrial dynamics is critical for infection. We have identified two never reported components participating in mitochondrial dynamics, e.g. septins and MIC10. We have deciphered that for the histone H3 deacetylation and the gene repression necessary for efficient infection, the deacetylase SIRT2 is dephosphorylated on Ser25, an event allowing its association to chromatin after its translocation to the nucleus. In addition, we have shown that the recently discovered modification of mRNA (N6-adenosine methylation) is in two different locations (i.e. intestine and liver) affected by the intestinal microbiota revealing a complex regulation induced by the gut microbiota.
Our project has led to a series of important findings which have been published in high impact journals such as Science, Nature Microbiology, Cell Host and Microbe, Nature Communications, PNAS, EMBO Reports, Cell Reports… and presented to many meetings in both Europe and the US (e.g. Gordon conferences, FEMS general meeting). We published several reviews in prestigious journals (Annual Reviews of Microbiology, Nature Microbiology Reviews…). Two patents have been deposited.

For our objective 1 which concerns the bacterium itself.
By using an N-terminomic approach to identify all the N-terminal peptides and all the start codons of the proteins encoded in the genome when bacteria are grown in three different conditions, we have discovered a never reported mini protein present in many firmicutes and showed how a bacterium by using this mini protein can relay an extracellular stress to the intracellular nanomachine called the stressosome and coordinate the expression of a series of genes involved in the stress response. In addition, we have determined the detailed structure of the Listeria stressosome.
We have identified the first secreted bacterial RNA binding protein which during infection binds and activates RIG-I and leads to the production of type I interferon.
We have identified in the Listeria strains which are responsible for epidemics, a bacteriocin which acts in the gut and favors infection. There are similar proteins in other bacteria, including streptococci. We have identified another bacteriocin which can target Prevotella copri in the gut and modulate infection.
We have discovered two previously unknown antibiotic resistance genes in Listeria, one of them is acting using a novel mechanism , i.e. ribosome splitting.

For our objective 2 which concerns the cell organelles during infection.
We have discovered that septins, (small GTPases able to form filaments and rings) play a critical role in mitochondrial dynamics. Mitochondrial dynamics is necessary for infection. Fragmentation is induced by the bacterial protein listeriolysin O (LLO). By analyzing the proteins present at the mitochondria during infection of cultures cells with the wild type or with the LLO mutant, we have discovered a role for Mic10, a subunit of the MICOS complex in the non-classical and Drp1-independent transient mitochondrial fragmentation occurring during infection.

For our objective 3 which concerns transcription in the infected host and the transcripts of the host.
We have discovered that for histone H3K18 deacetylation and the ensuing gene repression necessary for efficient infection, the deacetylase SIRT2 is dephosphorylated on Ser25, an event allowing its association to chromatin after its translocation to the nucleus. In addition, we have shown that the epitranscriptomic mark N6 adenosine methylation on intestinal mRNA, is affected by the intestinal microbiota. Moreover, the microbiota also affects the N6A methylation in the liver, revealing a new layer of regulation induced by the gut microbiota.
Structure of the stressosome