1. Research Plan Summary

Background
At the transition from monocellularity to multicellularity, eukaryotic organisms acquired NADPH oxidases (NOX) whose function is the generation of reactive oxygen species (ROS). Three different genes coding for distant NOX enzymes are found in primitive amoebae whereas up to seven genes have been identified in mammals. The first NOX enzyme to be studied in detail is the phagocyte NADPH oxidase NOX2, and humans lacking this enzyme are immuno-deficient. Thus, one of the key functions of NOX enzymes lies in the host defense: from direct killing of microorganisms to induction of signaling cascade in response to host pathogen interaction. Phagocytosis is a key function of eukaryotic cells, conserved from amoebae to mammals. Phagocytosis is the uptake of relative large particles, such as bacteria. In amoebae, phagocytosis serves the essential purpose of nutrition, while in professional animal phagocytes uptake of bacteria ultimately leads to their killing and to antigen presentation to cells of the adaptive immune system. However, many aspects of phagocytosis are shared, including signaling and killing mechanisms. But some bacteria have developed survival strategies to resist killing by phagocytes.

Working hypotheses
Many obligatory or facultative intracellular bacteria have specific mechanisms to survive and/or to modify the hostile environment of the phagosome and may become clinically relevant pathogens. An increasingly attractive concept is the idea that pathogenicity of many intracellular bacteria was not acquired during interaction with their mammalian host, but is rather a consequence of the interaction with ancient phagocytes, such as free-living amoebae.

Specific aims and experimental design
In this research module, we propose to study the role of NOX enzymes in phagocytic response and signaling to three different types of intracellular pathogens: mycobacteria, staphylococci and chlamydiae. We will investigate the interaction of these bacteria with "ancient phagocytes", using Dictyostelium discoideum as model organism, and with "contemporary phagocytes", taking mouse macrophages and dendritic cells as model cells. The involved research groups will provide the following tools and expertise: T. Soldati's group has expertise in work with mycobacteria and with Dictyostelium; his group will make use of Dictyostelium mutants deficient in one or several NOX enzymes. KH. Krause's group has expertise in work with NOX enzymes, ROS measurements, and with staphylococci; his group will provide NOX2-deficient mice and macrophages and dendritic cells derived thereof. G. Greub's group has expertise in work with Chlamydia-like organisms and free-living amoebae. His group will provide some axenized amoebae, including Acanthamoeba and Hartmanella species. Ph.D. candidate 1 will explore the role of ROS in mycobacterial infections. It will assesses whether Dictyostelium can produce measurable ROS and determine which NOX isoform is involved. In parallel, the various steps of the infection cycle in Dictyostelium mutants (single and multiple KOs) in NoxA, B and C, as well as p22Phox will be studied. Survival, proliferation and dissemination of M. marinum, M. smegmatis, M. avium, as well as M. bovis BCG, as well as various M. marinum mutants will be monitored. The analysis will be extended to phagocytes from wild-type and NOX2-deficient mice. Ph.D. candidate 2 will investigate the role of NOX enzymes in the survival of S. aureus in Dictyostelium, and in mouse phagocytes. In low ROS generation phagocytes, such as macrophages and dendritic cells, S. aureus are not killed: These cells might represent a reservoir for S. aureus survival, but ROS might also contribute to host-defense signalling and antigen presentation. Ph.D. candidate 3 will investigate the role of NOX enzymes on the survival and replication of Waddlia chondrophila in both macrophages (WT and NOX-/- macrophages) and Dictyostelium (WT and NOX mutants). Waddlia replicates efficiently within both macrophages and amoebae and represents thus a good model for cell biology investigations of the interactions of chlamydiae with phagocytes. Results obtained with this model pathogen will then be extended to investigate how two other chlamydiae (Parachlamydia and Chlamydia pneumoniae) survive to the phagocytes microbicidal machinery. Noteworthy, these three chlamydiae use completely different strategies to survive to macrophages: Waddlia recruit mitochondria and escape to endoplasmic reticulum vacuoles, Parachlamydia resist to degradation in the endocytic pathway by preventing the acquisition of lysosomal hydrolases, whereas Chlamydia exit the endocytic pathway by trafficking to Golgi-associated vacuoles. Thus, the specific role of NOX enzymes in each of the various chlamydial survival strategies will be characterized.