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

NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway

Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5′UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA expor

Leishmania braziliensis prostaglandin F2α synthase impacts host infection

BACKGROUND: Prostaglandins (PG) are lipid mediators derived from arachidonic acid metabolism. They are involved in cellular processes such as inflammation and tissue homeostasis. PG production is not restricted to multicellular organisms. Trypanosomatids also synthesize several metabolites of arachidonic acid. Nevertheless, their biological role in these early-branching parasites and their role in host-parasite interaction are not well elucidated. Prostaglandin F2α synthase (PGF2S) has been observed in the Leishmania braziliensis secreted proteome and in L. donovani extracellular vesicles. Furthermore, we previously reported a positive correlation between L. braziliensis PGF2S (LbrPGF2S) expression and pathogenicity in mice. METHODS: LbrPGF2S gene expression and PGF2α synthesis in promastigotes were detected and quantified by western blotting and EIA assay kit, respectively. To investigate LbrPGF2S localization in amastigotes during bone marrow-derived macrophage infection, parasites expressing mCherry-LbrPGF2S were generated and followed by time-lapse imaging for 48 h post-infection. PGF2S homolog sequences from Leishmania and humans were analyzed in silico using ClustalW on Geneious v6 and EMBOSS Needle. RESULTS: Leishmania braziliensis promastigotes synthesize prostaglandin F2α in the presence of arachidonic acid, with peak production in the stationary growth phase under heat stress. LbrPGF2S is a cytoplasmic protein enriched in the secretory site of the parasite cell body, the flagellar pocket. It is an enzyme constitutively expressed throughout promastigote development, but overexpression of LbrPGF2S leads to an increase of infectivity in vitro. The data suggest that LbrPGF2S may be released from intracellular amastigotes into the cytoplasm of bone marrow-derived macrophages over a 48-hour infection period, using time-lapse microscopy and mCherry-PGF2S (mChPGF2S)-expressing parasites. CONCLUSIONS: LbrPGF2S, a parasite-derived protein, is targeted to the host cell cytoplasm. The putative transfer of this enzyme, involved in pro-inflammatory lipid mediator synthesis, to the host cell suggests a potential role in host-parasite interaction and may partially explain the increased pathogenicity associated with overexpression of LbrPGF2S in L. braziliensis. Our data provide valuable insights to help understand the importance of parasite-derived lipid mediators in pathogenesis

Simultaneous two-color imaging in digital holographic microscopy

We demonstrate the use of two-color digital holographic microscopy (DHM) for imaging microbiological subjects. The use of two wavelengths significantly reduces artifacts present in the reconstructed data, allowing us to image weakly scattering objects in close proximity to strongly-scattering objects. We demonstrate this by reconstructing the shape of the flagellum of a single-cell eukaryotic parasite Leishmania mexicana in close proximity to a more strongly-scattering cell body. Our approach also yields a reduction of approximately one third in the axial position uncertainty when tracking the motion of swimming cells at low magnification, which we demonstrate with a sample of Escherichia coli bacteria mixed with polystyrene beads. The two-wavelength system that we describe introduces minimal additional complexity into the optical system, and provides significant benefits

Variable bites and dynamic populations : new insights in Leishmania transmission

Leishmaniasis is a neglected tropical disease which kills an estimated 50,000 people each year, with its deadly impact confined mainly to lower to middle income countries. Leishmania parasites are transmitted to human hosts by sand fly vectors during blood feeding. Recent experimental work shows that transmission is modulated by the patchy landscape of infection in the host's skin, and the parasite population dynamics within the vector. Here we assimilate these new findings into a simple probabilistic model for disease transmission which replicates recent experimental results, and assesses their relative importance. The results of subsequent simulations, describing random parasite uptake and dynamics across multiple blood meals, show that skin heterogeneity is important for transmission by short-lived flies, but that for longer-lived flies with multiple bites the population dynamics within the vector dominate transmission probability. Our results indicate that efforts to reduce fly lifespan beneath a threshold of around two weeks may be especially helpful in reducing disease transmission

Identification and stage-specific association with the translational apparatus of TbZFP3, a CCCH protein that promotes trypanosome life-cycle development

The post-transcriptional control of gene expression is becoming increasingly important in the understanding of regulated events in eukaryotic cells. The parasitic kinetoplastids have a unique reliance on such processes, because their genome is organized into polycistronic transcription units in which adjacent genes are not coordinately regulated. Indeed, the number of RNA-binding proteins predicted to be encoded in the genome of kinetoplastids is unusually large, invoking the presence of unique RNA regulators dedicated to gene expression in these evolutionarily ancient organisms. Here, we report that a small CCCH zinc finger protein, TbZFP3, enhances development between life-cycle stages in Trypanosoma brucei. Moreover, we demonstrate that this protein interacts both with the translational machinery and with other small CCCH proteins previously implicated in trypanosome developmental control. Antibodies to this protein also co-immunoprecipitate EP procyclin mRNA and encode the major surface antigen of insect forms of T. brucei. Strikingly, although TbZFP3 is constitutively expressed, it exhibits developmentally regulated association with polyribosomes, and mutational analysis demonstrates that this association is essential for the expression of phenotype. TbZFP3 is therefore a novel regulator of developmental events in kinetoplastids that acts at the level of the post-transcriptional control of gene expression

Arginine methyltransferases as regulators of RNA-binding protein activities in pathogenic Kinetoplastids

A large number of eukaryotic proteins are processed by single or combinatorial post-translational covalent modifications that may alter their activity, interactions and fate. The set of modifications of each protein may be considered a "regulatory code". Among the PTMs, arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), can affect how a protein interacts with other macromolecules such as nucleic acids or other proteins. In fact, many RNA-binding (RBPs) proteins are targets of PRMTs. The methylation status of RBPs may affect the expression of their bound RNAs and impact a diverse range of physiological and pathological cellular processes. Unlike most eukaryotes, Kinetoplastids have overwhelmingly intronless genes that are arranged within polycistronic units from which mature mRNAs are generated by trans-splicing. Gene expression in these organisms is thus highly dependent on post-transcriptional control, and therefore on the action of RBPs. These genetic features make trypanosomatids excellent models for the study of post-transcriptional regulation of gene expression. The roles of PRMTs in controlling the activity of RBPs in pathogenic kinetoplastids have now been studied for close to 2 decades with important advances achieved in recent years. These include the finding that about 10% of the Trypanosoma brucei proteome carries arginine methylation and that arginine methylation controls Leishmania:host interaction. Herein, we review how trypanosomatid PRMTs regulate the activity of RBPs, including by modulating interactions with RNA and/or protein complex formation, and discuss how this impacts cellular and biological processes. We further highlight unique structural features of trypanosomatid PRMTs and how it contributes to their singular functionality

The post-transcriptional trans-acting regulator, TbZFP3, co-ordinates transmission-stage enriched mRNAs in Trypanosoma brucei

Post-transcriptional gene regulation is essential to eukaryotic development. This is particularly emphasized in trypanosome parasites where genes are co-transcribed in polycistronic arrays but not necessarily co-regulated. The small CCCH protein, TbZFP3, has been identified as a trans-acting post-transcriptional regulator of Procyclin surface antigen expression in Trypanosoma brucei. To investigate the wider role of TbZFP3 in parasite transmission, a global analysis of associating transcripts was carried out. Examination of a subset of the selected transcripts revealed their increased abundance through mRNA stabilization upon TbZFP3 ectopic overexpression, dependent upon the integrity of the CCCH zinc finger domain. Reporter assays demonstrated that this regulation was mediated through 3′-UTR sequences for two target transcripts. Global developmental expression profiling of the cohort of TbZFP3-selected transcripts revealed their significant enrichment in transmissible stumpy forms of the parasite. This analysis of the specific mRNAs selected by the TbZFP3mRNP provides evidence for a developmental regulon with the potential to co-ordinate genes important in parasite transmission

PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites

RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation

High-speed, three-dimensional imaging reveals chemotactic behaviour specific to human-infective Leishmania parasites

Cellular motility is an ancient eukaryotic trait, ubiquitous across phyla with roles in predator avoidance, resource access, and competition. Flagellar motility is seen in various parasitic protozoans, and morphological changes in flagella during the parasite life cycle have been observed. We studied the impact of these changes on motility across life cycle stages, and how such changes might serve to facilitate human infection. We used holographic microscopy to image swimming cells of different Leishmania mexicana life cycle stages in three dimensions. We find that the human-infective (metacyclic promastigote) forms display 'run and tumble' behaviour in the absence of stimulus, reminiscent of bacterial motion, and that they specifically modify swimming direction and speed to target host immune cells in response to a macrophage-derived stimulus. Non-infective (procyclic promastigote) cells swim more slowly, along meandering helical paths. These findings demonstrate adaptation of swimming phenotype and chemotaxis towards human cells