Housekeeping by Leishmania.

The observation that differentiation of parasite life cycle stages involves structural and functional reorganisation is very familiar. A recent paper by Besteiro and colleagues provides an intriguing insight into cellular remodelling during differentiation. They show that it is essential for Leishmania to tidy up and put the house in order before it can differentiate to mammal-infective stages.

Introduction

Anyone who has had even a brief encounter with the world of parasites will have quickly realized that the intricacies of their life cycles are one of the most fascinating aspects of parasitology. The ‘what’ of parasite life cycles is well understood in most cases, and is a mainstay of parasitology teaching. However, the ‘how’ still remains largely mysterious. In a recent paper by Besteiro et al. [1], the role of two fundamental cell biological processes – endosomal sorting and autophagy – were examined in the life cycle progression of L. major, in particular the differentiation of the mammal-infective stages called metacyclic promastigotes (Box 1).

Investigating endosomes

The authors began by analysing the expression of L. major vacuolar protein sorting (VPS)4, a homologue of yeast VPS4 that is an ATPase catalysing the formation of late endosomes. They showed localization of L. major VPS4 consistent with an endosomal location. To investigate the function of VPS4, L. major expressing a dominant-negative mutant version (VPS4E235Q) were created, in which a crucial residue in the active site was changed, rendering the enzyme non-functional. These mutants prematurely entered the stationary phase in culture, were defective in endosomal sorting, and were also unable to differentiate into metacyclic promastigotes.

Links to autophagy

It has been known for some time that metacyclogenesis (Box 1) and subsequent transformation into amastigotes (Box 1) are both accompanied by large scale changes in protein expression (i.e. significant turnover of cellular proteins). In such situations, cells will often seek to conserve resources by exploiting the autophagy pathway. Therefore, the authors went on to study the functioning of autophagy in the VPS4E235Q mutants by examining the localisation of a GFP-tagged autophagosomal marker (the ubiquitin-like protein ATG8). The mutants possessed more autophagosomes under normal conditions, but these could not be processed properly, and, as a consequence, the mutants were found to be less able to survive nutrient deprivation than wild-type controls, in which autophagy was shown to peak at the initiation of metacyclogenesis. This is a significant finding considering the in vivo development of metacyclic promastigotes. The infective stages are the end product of a series of transformation and differentiation steps that occur in the sand fly, and involve at least four different life cycle stages [2]. By the time the metacyclic promastigotes begin to appear during the ‘sugarmeal phase’ of infection (when sand flies are feeding on plant sugars), there are only relatively limited energy and nutrient resources available, compared with the riches of the earlier ‘bloodmeal phase’. Also, the precursors of the metacyclic promastigotes, leptomonad forms, invest considerable resources in the biosynthesis of promastigote secretory gel (PSG), which is a key factor in promoting transmission [3]. Thus, the ability of metacyclic promastigotes to maximise the remaining available energy and nutrient resources by exploiting autophagy enables them to sit and wait for a longer period until the opportunity of transmission by sand fly bites arises.

Blocking autophagy

Finally, the authors of the paper went on to test directly the importance of the autophagy pathway by creating knockout mutants defective in ATG4 (a cysteine protease that processes ATG8 and is key to the formation of autophagosomes). As predicted, the ATG4 mutants were defective in processing autophagosomes, were vulnerable to starvation and failed to undergo metacyclogenesis, thus independently confirming the results obtained using the VPS4E235Q expressors. Again, it is interesting to relate these findings to the in vivo scenario. As the authors note, autophagy is often triggered by high population density, and this probably causes nutrient depletion itself. Mature transmissible infections in sand flies are characterized by extremely high parasite population densities, much higher than is ever achieved in vitro, because the parasites are packed into the PSG plug that occludes the anterior midgut [4]. The plug functions as a metacyclic promastigote factory, thus the conditions that are likely to trigger autophagy and the differentiation of metacyclic promastigotes are co-incident in vivo. This paper shows that autophagy is an essential prerequisite for metacyclogenesis, whereby the parasites put their own house in order, by removing unwanted organelles and macromolecular complexes, as part of their pre-adapted phenotype for invasion of the mammalian host.

One interesting area that we do not fully understand is what sets this train of events in motion in the first place – what are the signals that trigger metacyclogenesis? Possible signals are low environmental pH [5], low oxygen tension [6] and nutritional depletion of tetrahydrobiopterin [7]. Interestingly, these are all likely to be associated with high population densities. Therefore, it might be possible to use the markers described by Besteiro et al. to examine this important part of the molecular aspect of their life cycle. In conclusion, it is satisfying to note that, once again, the study of the kinetoplastids has not only revealed an interesting molecular aspect to their life cycle biology, but also provided another example of an excellent system for examining the function and interaction of basic cell biology processes in eukaryotes.