Defining the molecular steps in the interaction of pathogenic bacteria with alveolar macrophages, a key step in pathogenesis and disease.

We have recently demonstrated that bacteria, reported to be exclusive extracellular pathogens, undergo a phase of intracellular replication in a specific subset of macrophages prior to invasive infection. This interaction is mediated by the macrophage receptor CD169 (siglec1). Interaction with CD169 and uptake by CD169+ macrophages has been reported for a series of sialylated pathogens including Neisseria meningitidis, Campylobacter jejuni, Streptococcus agalactiae and enveloped viruses. Other bacteria with sialylated surface carbohydrates or proteins including Haemophilus influenzae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Klebsiella pneumoniae K1, Escherichia coli K1, and Streptococcus suis are highly likely to share the same or similar CD169-binding associated phenotypes. While uptake by CD169+ cells has been described, the occurrence of bacterial intracellular replication in CD169+ cells, the specific steps leading to this intracellular replication and the role of these events in the pathogenesis of infection, have only been discovered most recently by our group for Streptococcus pneumoniae.

The hypothesis at the basis of this project is that specific molecular events in the interaction between bacteria and CD169+ cells can guide optimisation of strategies for infection prevention. To test this hypothesis the project will address the following two objectives:
- Characterisation of the steps in the intracellular fate of different pathogens, including Haemophilus influenzae and Streptococcus suis in CD169+ macrophages
- Characterisation of the microbial factors involved in this interaction

In order to design a robust approach, the project will address these interactions starting with two model pathogens, the human Gram-negative pathogen Haemophilus influenzae and the porcine Gram-positive pathogen Streptococcus suis. The other species listed above are available in the lab and recorded on the HBA forms and will be utilised in the later phases of the project to test the broader validity of the findings. As main source of CD169+ cells, porcine alveolar and peri-follicular splenic macrophages will be used. The laboratory hosting the student has recently set up models for primary porcine splenic and alveolar macrophage cultures. These models will be exploited to investigate the specific steps in the interaction of the bacteria with CD169+ cells including phagocytosis, vacuole maturation and mechanism of cell death. The tools will include confocal microscopy, cytofluorimetry, cytokine assays, gene expression profiling and the testing of panels of bacterial mutants. To test the impact on the outcome of infection a limited series of experimental infection in murine models will be performed.
The training will range from confocal microscopy, cytofluorimetry, cell and organ culture, bacteriology, next generation sequence analysis, to animal handling. The overall aim of the CASE project is to provide the student with a multidisciplinary training program on a cutting edge scientific topic within a tight academic-industrial interaction.