PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection

Context and Background:

Intracellular pathogens, such as Legionella pneumophila, are among the major causes of disease and death. Pathogens hijack host cells trying to create an intracellular environment favourable for their replication. Host cells on other hand have to develop and refine defence mechanisms to beat the invaders to the punch and get rid of them before the infection spreads (Flannagan RS et al., 2009).

Phagocytosis plays a crucial role in the host-pathogen interaction as it represents the main mechanism of clearance and degradation of the not-self. For this reason, extensive research has been funnelled into this field to grasp the phases of phagosome maturation, in a journey that from the early step of ingestion ultimately leads to the digestion of the invader. Shortly after the internalisation of a particle, the phagosome is acidified and loaded with antimicrobial peptides and hydrolases, thereby creating a non-permissive environment for pathogen survival. The timing of the delivery of these components to the phagosome is strictly regulated and relies on the interaction with phosphoinositol lipids (PIPs) (Bohdanowicz M. et al., 2013). Therefore, understanding the mechanism of PIPs production and how this relates to phagosome maturation can be extremely beneficial and clinically relevant.

The preprint in brief:

The work of Buckley at al. underlines the importance of the lipid kinase PIKfyve in early-phagosome maturation and pathogen killing, using Dictyostelium as a model system. The results reported in this preprint highlight a role for PIKfyve in the delivery of the V-ATPase pump and hydrolytic enzymes to the phagosome, favouring the formation of an acidic environment hostile to bacteria. PIP production is therefore essential for shielding host cells from a wide array of microbes.

Key points:

Laboratory strains of Dictyostelium can either feed by micropinocytosis or by internalising bacteria. The group exploit this characteristic to study host-pathogen interaction and address how PIKfyve kinase activity favours the host. By analysing the ability of a PIKfyve mutant strain to reduce the turbidity of a bacterial suspension and to grow on bacteria, they quickly realised that PIKfyve mutants effectively uptook bacteria but their growth rate was reduced, meaning that one or more steps in phagosome maturation was altered. The work goes on taking a closer look at the process and analysing the rate of phagosome acidification.

They found that, in the absence of PIKfyve, acidification was much slower and not as efficient as in the control. This acidification defect was due to a much slower recruitment of the proton pump V-ATPase to the phagosome, as assessed by imaging the dynamics of GFP-tagged versions of the VTPase subunits VatM and VatB (GFP-VatM and GFP-VatB, respectively). In addition, PIKfyve mutant cells showed a lack of proteolitic activity despite the levels of hydrolytic enzymes, such as Cathepsin D being unchanged (if not higher) compared to control, suggesting again an improper delivery to the phagosome.

Given that acidification and proteolitic activity contribute to bacteria killing, Buckley at al. tested how PIKfyve defective cells performed when plated on layers of a panel of different non-pathogenic bacteria. PIKfyve mutants were unable to efficiently grow on any of the bacteria tested and phagocytosed bacteria survived more than 3 times longer than in the control. All together, these data suggest that PIKfyve has a general role in killing and digesting a broad range of microbes.

Is PIKfyve activity required to protect host cells from infection? To address this final but yet crucial point, PIKfyve mutant cells were grown in the presence of the opportunistic human pathogen Legionella pneumophila. As expected, bacteria uptake was unperturbed, but again, the invaders survived much longer in PIKfyve mutant cells. Most importantly, the group proved that the pathogen grew much more rapidly and to a much greater extent in cells lacking PIKfyve activity than in control, meaning that in the absence of PIKfyve Legionella stands a much better chance of creating a permissive environment for its own replication.

Relevance:

Phagosome maturation requires the sequential fusion with several membrane compartments until the formation of the phagolysosome, responsible for the final degradation of the invader. Several pathogens therefore have developed the ability to alter the composition and levels of phosphoinositol lipids on the phagosome membrane to avoid its fusion with the lysosome, thereby escaping digestion. Understanding how PIP levels are controlled is therefore clinically relevant and important to get a better understanding of host-pathogen interaction.

Questions to the authors:

• Is the mechanism of how bacteria manipulate PIP levels known? Could they achieve this by inactivating PIKfyve kinase?
• SNAREs and Rabs are important for membrane fusion. Is their localisation to phagosomes altered in PIKfyve-null cells? Is there any specific SNARE/Rab affected?
• In Drosophila, highly vacuolated macrophages are dramatically impaired in their migration (Evans I. et al. 2013). Given that PIKfyve-null cells are highly vacuolated and the enzyme evolutionary conserved, can you predict whether, as a secondary effect, the inhibition of the kinase would impair the patrolling activity of immune cells?

References:

• Flannagan RS, Cosio G, Grinstein S. Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nat Rev Microbiol. 2009;7(5):355-66. Epub 2009/04/17.
• Bohdanowicz M, Grinstein S. Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis. Physiological reviews. 2013; 93(1): 69-106.
• Evans IR, Ghai PA, Urbančič V, Tan KL, Wood W. SCAR/WAVE-mediated processing of engulfed apoptotic corpses is essential for effective macrophage migration in Drosophila. Cell Death Differ. 2013 May; 20 (5): 709-20.

Posted on: 13 September 2018 , updated on: 17 September 2018

doi: https://doi.org/10.1242/prelights.4773

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