Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival.

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.

Developmental differentiation in Leishmania lifecycle progression: post-transcriptional control conducts the orchestra.

The successful progression of Leishmania spp. through their lifecycle entails a series of differentiation processes; the proliferative procyclic promastigote forms become quiescent, human-infective metacyclic promastigotes during metacyclogenesis in the sandfly vector, which then differentiate into amastigotes during amastigogenesis in the mammalian host. The progression to these infective forms requires two components: environmental cues and a coordinated cellular response. Recent studies have shown that the Leishmania cellular transformation into mammalian-infective stages is triggered by broad changes in the absolute and relative RNA and protein levels. In this review, we will discuss the implications of Leishmania transcriptomic and proteomic fluctuations, which adapt the parasitic cell for survival.

Investigating the Swimming of Microbial Pathogens Using Digital Holography.

To understand much of the behaviour of microbial pathogens, it is necessary to image living cells, their interactions with each other and with host cells. Species such as Escherichia coli are difficult subjects to image: they are typically microscopic, colourless and transparent. Traditional cell visualisation techniques such as fluorescent tagging or phase-contrast microscopy give excellent information on cell behaviour in two dimensions, but no information about cells moving in three dimensions. We review the use of digital holographic microscopy for three-dimensional imaging at high speeds, and demonstrate its use for capturing the shape and swimming behaviour of three important model pathogens: E. coli, Plasmodium spp. and Leishmania spp.