Regulation of endosomal and phagosomal transport
Kuijl, C.P.
Citation
Kuijl, C. P. (2008, October 15). Regulation of endosomal and phagosomal transport.
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Summary
Nederlandse samenvatting
Publications
Curriculum vitae
133 summary and discussion
Summary and discussion
Pathogenic intracellular pathogens have co-evolved with humans. By hijacking and manipulating the normal functions of host cells, these bacteria are able to create a niche where they can survive and replicate. The endocytic pathway is used and modified by bacteria such as Salmonella typhimurium (Salmonella) and Mycobacterium tuberculo- sis. We attempt to acquire knowledge on the normal function of the endocytic pathway, by investigating how pathogenic bacteria manipulate this pathway.
We have taken Salmonella as model sys- tem since it traverses the endocytic route in a method similar but not identical to normal endocytosis. Salmonella contains a type III secretion system which injects proteins into the host cells to ensure entry into the host cell via the endocytic pathway. These inject- ed proteins modify the endocytic route such that Salmonella can survive inside the host cell. Normally the endocytic pathway starts with pH neutral vesicles (early endosomes) which become acidified during maturation (late endosomes and finally lysosomes).
This acidification leads to activation of lu- minal proteases belonging to the cathepsin family and degradation of the content of ly- sosomes. To survive inside the cell, Salmo- nella actively prevents the maturation of the vesicles it resides in (the phagosome) into ly- sosomes. Salmonella prevents fusion of the phagosome with lysosomes containing acti- vated cathepsins. In Chapter 2 we describe how the Rab7 Interacting Lysosomal Pro- tein (RILP) stimulates transport and fusion of phagosomes with lysosomes, resulting in a hostile environment in which Salmonella cannot replicate.
Salmonella could serve as an exogenous source of antigens for MHC class II mol- ecules, since MHC class II molecules are loaded with foreign antigens in specialized lysosomes called MHC class II-contain- ing compartments (MIICs). These MIICs
can me multi-vesicular or multi-lamellar containing immune molecules on both the limiting membrane and the inner membrane structures. In chapter 3 the interaction be- tween two immune molecules in the MIIC is described in great detail. One molecule is HLA-DR, the MHC class II molecule that presents extracellular (foreign) derived anti- gens to CD4 positive T-cells of the immune system to elicit the production of specific an- tibodies. The other is HLA-DM which facil- itates the exchange of the self peptide CLIP with a potential antigenic peptide derived from an exogenous source (e.g. Salmonella).
Both HLA-DR and HLA-DM are present on the limiting membrane of a Salmonella con- taining phagosomes. We show that HLA- DM facilitated peptide exchange takes place on the vesicles inside the MIICs but not on the limiting membrane of this compartment.
Since there are little to no internal vesicles present in the phagosome, loading of MHC class II molecules is impaired. This could be a mechanism of salmonella to evade the im- mune system.
The endocytic route is well studied in cell biology. Both the MIIC and the Salmonella phagosome are positive for the small GT- Pase Rab7. Rab7 can recruit RILP to induce transport and fusion (chapter 2). How these transport and fusion events are regulated are unknown and are the subject of research in chapter 4. Small GTPases cycle between a membrane active form (GTP bound) and a membrane dissociated form (GDP bound).
The hydrolysis of GTP to GDP might there- fore function as a molecular switch to stop transport and fusion of the Rab7 positive compartment. GTPase Activating Proteins (GAPs) can stimulate the GTPase activity of small GTPases and might in this way switch of transport and fusion. RILP reduces the intrinsic GTPase activity of Rab7 and more- over sterically hinders binding of the iden- tified Rab7GAP (TBC1D15) to Rab7, sug- gesting that transport and fusion events are not regulated by GAPs. We show that Rab7
summary and discussion
mediated motor recruitment is depended on RILP, but transport is only initiated when ORP1L and RILP are present. ORP1L is a cholesterol sensing protein and like RILP binds Rab7 in its GTP bound form. The cho- lesterol sensing domain of ORP1L is essen- tial for keeping the molecules in a conforma- tion allowing for transport when bound to cholesterol. When this domain is removed no dynein mediated transport takes place.
When vesicles have low cholesterol content or when the ORD domain is removed, the ORP1L molecules exposes a domain that re- cruits yet another molecule called VAP-A.
This VAP-A prevents binding of the dynein motor to the Rab7-RILP-ORP1L complex.
The Niemann-Pick disease (caused by muta- tions in the NPC1 gene) causes cholesterol accumulation in lysosomes and is charac- terized by clustering of these vesicles in the perinuclear area. When ORP1L missing the cholesterol sensing domain is expressed in cells missing the NPC1 gene (normally re- sulting in the Niemann-Pick phenotype), vesicles are cholesterol laden but scattered.
Dynein mediated transport of late endo- somes and lysosomes seems therefore de- pendent on the cholesterol contents of these vesicles.
Many technically challenging and power- ful techniques in this thesis are used to in- vestigate a variety of biological questions. I would like to draw special attention to the siRNA mediated screening used in chap- ter 6. When large siRNA libraries are used and/or the scientific question is not specific enough, an overwhelming number of ‘hits’
will be found. But this technique can be ex- tremely powerful and can quickly lead to results when small dedicated libraries are used in combination with a specific scientif- ic question. For instance, in chapter 6 ‘only’
the knockdown of kinases was tested for the effect on intracellular Salmonella growth.
The ‘hits’ from this screen however do not explain the biological effect observed.
described in all chapters remains therefore an essential activity to move from a list of genes to specific biological functions for those genes.
While chapter 2 and 3 describe the ef- fect of Salmonella on known pathways in the host cell, chapter 6 identifies novel host proteins whose activities are modified or needed by Salmonella and Tuberculosis.
Salmonella and Tuberculosis manipulate transport within the host cell. Since trans- port is amongst others regulated by kinases and essential for pathogen survival, we in- vestigated whether kinase inhibitors could have any effect on Salmonella and Tuber- culosis survival inside the host cell. The Protein Kinase A (PKA) inhibitor H-89 had a profound effect on Salmonella and Tuber- culosis proliferation inside the host cell, the application of H-89 prohibited any replica- tion of Salmonella and Tuberculosis. Other inhibitors of PKA and siRNA mediated PKA knockdown did not result in this block in proliferation, suggesting that not PKA but another kinase was targeted by the H- 89 compound. A subsequent siRNA screen targeting kinases and kinase related proteins identified 10 kinases involved in Salmonella replications. Derivatives of H-89 were made and the in vivo inhibitory effect on Salmo- nella growth was compared to the in vitro inhibitory effect on kinase activity. Only the Protein Kinase B (PKB/Akt) in vitro profile matched the in vivo profile, indicating that not PKA but PKB is the relevant target of H- 89 in the case of intracellular block of Sal- monella replication. The activation of PKB/
Akt by Salmonella regulates actin dynamics via a protein called PAK4 and transport/fu- sion via the small GTPase Rab14. Activat- ing PAK4 or Rab14 independent of PKB/Akt can therefore (partially) attenuate the bacte- ricidal effect of PKB/Akt inhibitors.
Surprisingly, the genes PKB/Akt and PAK4 that have been identified to be essen- tial for Salmonella survival inside the host
135 summary and discussion
human cancers. It is therefore tempting to speculate that the genes activated by patho- gens such as Tuberculosis and Salmonella are similar to the genes activated in cancer.
The activation of these genes promotes sur- vival of these cells and at the same time sur- vival of the pathogens in these cells. Com- pounds discovered as antibiotics are now used as anti cancer drugs. The current anti cancer drugs could in a similar fashion pro- vide us with a novel class of antibiotics to treat pathogens resistant all current ‘classic’
antibiotics.
