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Membrane dynamics in host – pathogen interactions

This research line is embedded in the Research programs Strategic Infection Biology of the Faculty of Veterinary Medicine and in Infection & Immunity of Utrecht University.

After binding to plasma membrane, pathogens are taken up by phagocytosis or a related process. Upon entry in the cell, pathogens often secrete effectors into the host cell, modulating cellular responses in an attempt to escape degradation by lysosomes. Host cell targets include enzymes regulating membrane dynamics and host cell lipid metabolism.

 

Host cell membrane dynamics during Salmonella infection

Salmonella typhimurium translocates effector proteins into host cells. These effectors influence the host cell to allow invasion, survival and replication inside phagosomal compartments termed Salmonella-containing vacuoles (SCVs). We investigate several of these effectors with respect to their effect on subcellular SCV localization and effects on lipid metabolism.

We developed a SILAC-based quantitative proteomics approach to investigate the interactions of Salmonella typhimurium with the secretory pathway in human epithelial cells (Vogels et al (2011) Proteomics). 105 proteins were shown to be up- or down regulated upon Salmonella infection. The functional relevance of several of the identified proteins was confirmed by siRNA-mediated depletion of affected proteins. Surprisingly, depletion of some of th4ese effector proteins such as Stomatin resulted in a dispersal of intracellular Salmonella microcolonies (Figure). The relevance of this observation is under current investigations.

Salmonella Golgi Merge
Path Hela siRNA 1 Control siRNA
Path Hela siRNA 2 Stomatin siRNA
Figure: HeLa cells were transfected with control or siRNA oligos targeting Stomatin and infected with wt Salmonella for 6 hours. In green is shown the LPS staining, identifying the Salmonella microcolony, and in red the GM130 (Golgi) staining.

Lipid turnover during phagosomal maturation

Little is known about the role of lipids in phagocytosis and maturation. We use a well established assay to study changes in the lipidome of phagosomes during maturation. Using a in house lipidomics facility, we are currently characterizing the kinetics of lipid turnover (>1000 lipids) during phagosomal maturation. Surprisingly, every time point of maturation is characterized by a unique lipid composition (Figure). A long term goal is to understand the interference of pathogens with host cell lipid metabolism and how pathogens interfere with the phagosomal maturation process to avoid their degradation (van Meer et al (2010) Progress Lipid Res).

Path Pago lipid PCA 3
Figure: Phospholipid turnover during phagosomal maturation, and identification of marker lipids by discriminant analysis. In left panel, the score plot again shows distinct lipid compositions at different stages of phagosome maturation. Right panel: loadings of individual lipid species.

GAPR-1 at lipid-enriched microdomains of the Golgi complex

We previously isolated Golgi-derived lipid-enriched microdomains (also termed lipid rafts) with a unique subset of proteins and lipids (Gkantiragas et al (2001) Mol. Biol. Cell). Changing the properties of lipid rafts affects the intra-Golgi membrane fusion reaction (Stueven et al (2003 J. Biol. Chem). Another raft protein is involved in maintenance of the structure of the Golgi complex (Li et al. (2007) Mol. Biol. Cell). We also characterized a previously unknown protein in Golgi-derived lipid rafts named GAPR-1 (Eberle et al (2002) J. Cell Sci; Serrano et al (2004) J. Mol. Biol.). GAPR-1 belongs to the superfamily of plant pathogenesis-related (PR-1) proteins that play an essential role in the defence of plants. The plant defence system is reminiscent of the innate immune system in mammals. Thus, the discovery of GAPR-1 suggests a role in immune defence for lipid rafts at the Golgi complex.

We recently found evidence for a novel dimeric structure of GAPR-1 in which one of the monomeric subunits of the crystallographic dimer is rotated by 28.5° (figure). Our combined data suggest that the charge properties of the lipid bilayer can regulate GAPR-1 dynamics as a potential mechanism to modulate GAPR-1 function. This knowledge is applied to investigate changes in membrane dynamics in host cells during host-pathogen interactions.

Path GAPR 4
Figure: The monomeric structures are superimposed using residues 4-152. The left monomers (yellow) are positioned in the same orientation to allow visualization of the rotation of the partner monomers at the right (partner monomer in red). 

Lipid droplets in host-pathogen interactions

Toxoplasma gondii evade the host immune response by adopting an intracellular lifestyle residing in modified intracellular vacuoles that are not targeted for phago-lysosomal destruction. To scavenge nutrients from the host, lipids and even whole host organelles like lipid droplets (LDs) can be translocated across pathogen-containing vacuoles. Immunity-related GTPases (IRGs) are essential factors in cell-autonomous resistance against vacuolar pathogens like Toxoplasma gondii and recently, a member of the IRG family, Irgd (IRG-47), has been identified as potential interaction partner of the lipid droplet (LD)-associated protein ADRP (adipose differentiation-related protein). We confirmed the presence of IRG proteins on lipid droplets (Figure). Using this model system, we study the involvement of IRG proteins and lipid droplets in host-pathogen interactions.

IRG TIP47 LDR Phase
Path TOX IRG 5 Path TOX TIP47 6 Path TOX LDR 7 Path TOX Phase 8
Figure: IRG localises to LDs stained by a neutral lipid dye (LDR) and TIP47 in IFNγ-induced cells.