Toxins antitoxin systems are present in the genome of many pathogenic bacteria and understand their roles in the mechanisms of persistence is a major challenge to understand the infections with these bacteria.
H. pylori chronically colonizes the stomach of half of the world's human population. This infection is the major cause of gastric diseases such as peptic ulcers or adenocarcinoma that causes annually the death of 800,000 people worldwide. Almost all bacterial genomes, including those of pathogens, contain a very large number of toxin-antitoxin operons (TA). These toxins target cellular functions to regulate the growth and death of bacteria. Present hypotheses suggest a role in growth regulation and programmed cell death in response to stress conditions (such as nutrient starvation, high temperatures, oxidative stress, or exposure to antibiotics). In BacTox1 project, we propose to study the role of a TA system recently identified in H. pylori. The aim of our project is to show that the toxin plays a role in the formation of persister cells in this bacterium. These cells, which are present in very small numbers in a normal population, are dormant and are thus recalcitrant to antibiotic treatment.
This project uses multidisciplinary approaches ranging from molecular biology to electron microscopy, through structural biology approaches (nuclear magnetic resonance spectroscopy, NMR).
Bactox1 focuses on the study of the expression of TA system in H. pylori. We have shown that expression of the toxin causes the death of the bacterium resulting in a morphological change of the cells. We characterized this toxin which is a small peptide which interacts with the inner bacterial membrane and causes the formation of the so-called rod-shaped cells named «coccoides«. These cells were also observed in the stomach of patients infected by H. pylori. The TA recently discovered in this bacterium is one of the major molecular mechanisms established by the bacterium to infect and persist in the human stomach.
Research on TA systems began only recently, and many questions remain unanswered. It is likely that new TA systems will be discovered in the future. Deciphering the mechanisms of their toxic activities offer new perspectives on the diverse roles these systems may play in the physiology of pathogenic bacteria and in the mechanisms of persistence in response to antibiotic treatments. Another interesting perspective is that the toxins of these systems can be used in turn to develop new antibiotics.
Two articles are in preparation :
Mechanistic insights in type I toxin antitoxin regulation in Helicobacter pylori.
Hélène Arnion, Lamya EL Mortaji, Sandrine Chabas, Isabelle Iost, Jérémy Reignier, Hilde de Reuse and Fabien Darfeuille
This article focuses on the study of the expression of AapA1/IsoA1 toxin antitoxin system in Helicobacter pylori. We have shown that expression of the peptide is toxic for the bacterium leading to a strong morphological change. Indeed in response to the toxin expression, H. pylori passes from the spiral to the coccoid form.
Structural characterization of the Helicobacter pylori AapA1 toxin
Dursun Nizam Korkut, Lamya El Mortaji, Sandrine Chabas, Arnion Hélène, Alves Isabelle, Hilde de Reuse, Fabien Darfeuille and Gilmar Salgado
In this study we have shown that the toxin interacts strongly with the inner membrane of H. pylori. The NMR spectroscopy study of the peptide indicates that a 19 amino acid domain folds into an alpha helix which could be inserted into the membrane. A mutant of the peptide whose expression is not toxic to the bacterium, has a different conformational dynamics in this region.
Almost all free-living bacterial genomes, including those of pathogens, contain a large number of toxin-antitoxin (TA) operons. These toxins target various cellular functions to regulate growth and death of bacteria. Although these systems were initially identified as playing a key role in the plasmid maintenance during the bacterial cell division, the function of these systems when they are present on the chromosome remains enigmatic. The toxin-antitoxin systems are hypothesized to induce a reversible growth arrest or a programmed cell death, depending on the stress conditions (eg, starvation, high temperature, oxidative stress, or exposure to antibiotics). In the BacTox1 project, we propose to study the role of a chromosomal toxin-antitoxin cassette recently identified in the human pathogen Helicobacter pylori (Hp). Our preliminary results show that this module consists of two genes aapA1 and isoA1, which are expressed in reverse orientation. The AapA1 gene encodes a small peptide of 30 amino acids whose expression is toxic to the cells. This expression is repressed by the antisense RNA (IsoA1) that plays the role of an antitoxin by preventing the synthesis of the A1 toxin. The aim of our project is to show that this toxin plays a role in the formation of persister cells. Indeed it appears that some of these TA systems when expressed stochastically are able to induce the formation of persistent cells. These cells, which are present in very small numbers in a normal population, are in a state of dormancy and are recalcitrant to antibiotic treatments. To investigate whether the A1 toxin is expressed in a subpopulation of dormant cells, we propose to develop several tools allowing its detection in living cells. During this project we also propose to identify partners of the A1 toxin and to determine its NMR solution structure. We believe that the tools developed in this proposal will help understanding the mode of action of this type of toxin in the regulation of bacterial growth. Research on chromosomally-encoded TA systems has started only recently, and many questions remain to be answered. It is likely that new TA systems will be discovered, and deciphering the mechanisms of their toxic functions will provide new insights into the highly diverse roles of TA systems in the cell physiology of bacteria.
Monsieur Fabien Darfeuille (INSERM U869,) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
ARNA laboratory INSERM U869
ARNA laboratory INSERM U869,
IP Institut Pasteur, Unité Pathogenèse de Helicobacter
Help of the ANR 340,000 euros
Beginning and duration of the scientific project: February 2013 - 36 Months