Research group of D. Soulat, Ph.D.

Microbiology

Head of Institute:
Prof. Dr. med. Christian Bogdan

Study of new virulence factor of Listeria monocytogenes

How do phosphatases secreted by pathogens interfere with immune response during infection ?

Infectious microorganisms have developed many ways to drive the host-pathogen interaction during the infection process. Among this large arsenal of virulence factor, the secretion of phosphatase by pathogen appears to be one of the most efficient tactics to interfere with the host cell response. Indeed, the reaction of phosphorylation/dephosphorylation of protein and lipids is the most common modification used by cell to control their cellular machinery.

The laboratory is therefore focusing its research on newly discovered phosphatases secreted by two distinct pathogens: (1) the bacteria Listeria monocytogenes and (2) the parasite Leishmania major.

1. Mechanism of action of the atypic phosphatase LipA secreted by L. monocytogenes

LipA is a new virulence factor recently discovered in the intracellular Gram-positive bacteria Listeria monocytogenes, the causative agent of Listeriosis. This Food-borne disease is rare but can cause clinically serious symptoms with a high case fatality rate in immuno-compromised individuals. After ingestion of contaminated food, L. monocytogenes crosses the epithelial barrier of the intestine and disseminates in deeper tissues via the lymphatics and the blood stream. At first, spleen and liver are infected. There, the bacterium replicates and uses these organs as reservoir for further spreading in the brain and the placenta. This pathogenicity relies on an elaborated arsenal of virulence factors.

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Legend: Listeria cell cycle (adapted from Tilney and Portnoy, 1989)

We recently identified a new virulence factor of L. monocytogenes , the secreted phosphatase LipA (Kastner et al. 2011). This enzyme exhibits unusual features such as tyrosine as well as phosphoinositol-phosphate (PIP) phosphatase activity in vitro. Strikingly, bacteria lacking LipA are severely attenuated in virulence in vivo independently of the infection route, which goes in line with its expression pattern strictly dependent on the entry of the bacteria in the blood stream.

Legend: Role of LipA for the virulence of L. monocytogenes in mice.

To control the infection by L. monocytogenes, organism strongly relies on the early innate immunity, while adaptive immunity such as cytotoxic T cell is required for the final clearance. Neutrophils are critical players during the early phase of Listeria infection (14). They are among the first cells to phagocyte bacteria and kill them via the production of reactive oxygen species (ROS). This bactericidal activity is based on the NADPH oxidase activation and assembly at the neutrophil membrane. This process is tightly regulated by multiple receptors controlling through the transient synthesis of PIPs.

We discovered the in vitro specificity of the Listeria phosphatase LipA and we showed its involvement in the virulence of the bacteria. However, the cellular and molecular targets of this phosphatase still need to be discovered.

Aim of the project

  1. The determination of the innate immune cells affected by LipA. We are analyzing ex vivo and in vivo the effect of LipA on the activation and function of different immune cells, starting with neutrophils and blood monocyte derived cells.
  2. The identification of LipA substrate. We are looking at both side of the LipA specificity. (a) A intracellular study of LipA behavior is carried out to determine the PIP specificity. (b) A substrate trapping for LipA is also in progress to identify putative phosphoprotein substrates. 

2. Effect of secreted phosphatases of Leishmania parasites on the immune systems of infected host

Leishmania major is the causative agent of cutaneous leishmaniasis, a worldwide prevalent parasitic disease transmitted by sand fly bite to human. In its insect vector, the parasite resides in an extracellular, highly infective, promastigote form. Once in its mammal host, the parasite enters myeloid host cells with a high prevalence for macrophages. There, they transform into intracellular amastigotes, which facilitate the spread in the host organism. However, only few Leishmania virulence factors have been characterized to date.

Our laboratory identified two putative new virulence factor produced by Leishmania parasites (named Lp1 and Lp4). These two homologous tyrosine phosphatases were shown to be partly secreted by Leishmania major via the exosome pathway. Interestingly, these phosphatases have a strong structural homology with the human phosphatase PRL-1 (Phosphatase of Regenerating Liver 1) that is involved in the regulation of numerous cellular features such as growth and cell motility. Based on this homology, we hypothesized that the Leishmania phosphatases could interfere with the PRL-1 function to modulate the host response during infection.

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Legend: Heterotrimeric model of Lp1 (green) and PRL-1 (red) realized from the crystal structure of the homotrimer of PRL-1 and the crystal structure of Lp1 (Prof. Sticht, FAU Erlangen). Lp1, like PRL-1 could be localized at the membrane thanks to a positive stretch at its C-terminal end and a Farnesylation site not depicted here. By interacting with PRL-1, Lp1 could interfere with the regular activity of these enzymatic complex or change its substrate specificity.

Aim of the Project

  1. The in vitro characterization of these enzymes is allowing us to determine their range of substrate specificity in vivo. The study of their subcellular localization also helps us to determine their biological function.
  2. Ectopic expression and deletion of the genes in Lesihmania parasite is also carried out to define their function during the infection course in a mouse model of cutaneous leishmaniasis.
 
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Summary