Bloodstream form pre-adaptation to the tsetse fly in Trypanosoma brucei

African trypanosomes are sustained in the bloodstream of their mammalian hosts by their extreme capacity for antigenic variation. However, for life cycle progression, trypanosomes also must generate transmission stages called stumpy forms that are pre-adapted to survive when taken up during the bloodmeal of the disease vector, tsetse flies. These stumpy forms are rather different to the proliferative slender forms that maintain the bloodstream parasitaemia. Firstly, they are non proliferative and morphologically distinct, secondly, they show particular sensitivity to environmental cues that signal entry to the tsetse fly and, thirdly, they are relatively robust such that they survive the changes in temperature, pH and proteolytic environment encountered within the tsetse midgut. These characteristics require regulated changes in gene expression to pre-adapt the parasite and the use of environmental sensing mechanisms, both of which allow the rapid initiation of differentiation to tsetse midgut procyclic forms upon transmission. Interestingly, the generation of stumpy forms is also regulated and periodic in the mammalian blood, this being governed by a density-sensing mechanism whereby a parasite-derived signal drives cell cycle arrest and cellular development both to optimise transmission and to prevent uncontrolled parasite multiplication overwhelming the host.In this review we detail recent developments in our understanding of the molecular mechanisms that underpin the production of stumpy forms in the mammalian bloodstream and their signal perception pathways both in the mammalian bloodstream and upon entry into the tsetse fly. These discoveries are discussed in the context of conserved eukaryotic signalling and differentiation mechanisms. Further, their potential to act as targets for therapeutic strategies that disrupt parasite development either in the mammalian bloodstream or upon their transmission to tsetse flies is also discussed

Assembling the components of the quorum sensing pathway in African trypanosomes

African trypanosomes, parasites that cause human sleeping sickness, undergo a density-dependent differentiation in the bloodstream of their mammalian hosts. This process is driven by a released parasite-derived factor that causes parasites to accumulate in G1 and become quiescent. This is accompanied by morphological transformation to ‘stumpy’ forms that are adapted to survival and further development when taken up in the blood meal of tsetse flies, the vector for trypanosomiasis. Although the soluble signal driving differentiation to stumpy forms is unidentified, a recent genome-wide RNAi screen identified many of the intracellular signalling and effector molecules required for the response to this signal. These resemble components of nutritional starvation and quiescence pathways in other eukaryotes, suggesting that parasite development shares similarities with the adaptive quiescence of organisms such as yeasts and Dictyostelium in response to nutritional starvation and stress. Here, the trypanosome signalling pathway is discussed in the context of these conserved pathways and the possible contributions of opposing ‘slender retainer’ and ‘stumpy inducer’ arms described. As evolutionarily highly divergent eukaryotes, the organisation and conservation of this developmental pathway can provide insight into the developmental cycle of other protozoan parasites, as well as the adaptive and programmed developmental responses of all eukaryotic cells

Non-linear hierarchy of the quorum sensing signalling pathway in bloodstream form African trypanosomes.

Trypanosoma brucei, the agents of African trypanosomiasis, undergo density-dependent differentiation in the mammalian bloodstream to prepare for transmission by tsetse flies. This involves the generation of cell-cycle arrested, quiescent, stumpy forms from proliferative slender forms. The signalling pathway responsible for the quorum sensing response has been catalogued using a genome-wide selective screen, providing a compendium of signalling protein kinases phosphatases, RNA binding proteins and hypothetical proteins. However, the ordering of these components is unknown. To piece together these components to provide a description of how stumpy formation arises we have used an extragenic suppression approach. This exploited a combinatorial gene knockout and overexpression strategy to assess whether the loss of developmental competence in null mutants of pathway components could be compensated by ectopic expression of other components. We have created null mutants for three genes in the stumpy induction factor signalling pathway (RBP7, YAK, MEKK1) and evaluated complementation by expression of RBP7, NEK17, PP1-6, or inducible gene silencing of the proposed differentiation inhibitor TbTOR4. This indicated that the signalling pathway is non-linear. Phosphoproteomic analysis focused on one pathway component, a putative MEKK, identified molecules with altered expression and phosphorylation profiles in MEKK1 null mutants, including another component in the pathway, NEK17. Our data provide a first molecular dissection of multiple components in a signal transduction cascade in trypanosomes

Plant-like phosphofructokinase from Plasmodium falciparum belongs to a novel class of ATP-dependent enzymes

Malaria parasite-infected erythrocytes exhibit enhanced glucose utilisation and 6-phospho-1-fructokinase (PFK) is a key enzyme in glycolysis. Here we present the characterisation of PFK from the human malaria parasite Plasmodium falciparum. Of the two putative PFK genes on chromosome 9 (PfPFK9) and 11 (PfPFK11), only the PfPFK9 gene appeared to possess all the catalytic features appropriate for PFK activity. The deduced PfPFK proteins contain domains homologous to the plant-like pyrophosphate (PPi)-dependent PFK β and α subunits, which are quite different from the human erythrocyte PFK protein. The PfPFK9 gene β and α regions were cloned and expressed as His6- and GST-tagged proteins in Escherichia coli. Complementation of PFK-deficient E. coli and activity analysis of purified recombinant proteins confirmed that PfPFK9β possessed catalytic activity. Monoclonal antibodies against the recombinant β protein confirmed that the PfPFK9 protein has β and α domains fused into a 200 kDa protein, as opposed to the independent subunits found in plants. Despite an overall structural similarity to plant PPi-PFKs, the recombinant protein and the parasite extract exhibited only ATP-dependent enzyme activity, and none with PPi. Unlike host PFK, the Plasmodium PFK was insensitive to fructose-2,6-bisphosphate (F-2,6-bP), phosphoenolpyruvate (PEP) and citrate. A comparison of the deduced PFK proteins from several protozoan PFK genome databases implicates a unique class of ATP-dependent PFK present amongst the apicomplexan protozoans

Genome-wide dissection of the quorum sensing signalling pathway in <em>Trypanosoma brucei</em>

The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ‘slender’ forms and arrested ‘stumpy’ forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing (QS) in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission(1). This response is triggered by an elusive ‘stumpy induction factor’ (SIF) whose intracellular signaling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently to SIF but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPTcAMP) or 8-pCPT-2′-O-Me-5′-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide ion torrent-based RNA interference Target sequencing identified cohorts of genes implicated in each step of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo, whilst the putative RNA-binding protein, RBP7, was required for normal QS and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that QS signaling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for QS-interference based therapeutics

An organelle-tethering mechanism couples flagellation to cell division in bacteria

In some free-living and pathogenic bacteria, problems in the synthesis and assembly of early flagellar components can cause cell-division defects. However, the mechanism that couples cell division with the flagellar biogenesis has remained elusive. Herein, we discover the regulator MadA that controls transcription of flagellar and cell-division genes in Caulobacter crescentus. We demonstrate that MadA, a small soluble protein, binds the type III export component FlhA to promote activation of FliX, which in turn is required to license the conserved σ54-dependent transcriptional activator FlbD. While in the absence of MadA, FliX and FlbD activation is crippled, bypass mutations in FlhA restore flagellar biogenesis and cell division. Furthermore, we demonstrate that MadA safeguards the divisome stoichiometry to license cell division. We propose that MadA has a sentinel-type function that senses an early flagellar biogenesis event and, through cell-division control, ensures that a flagellated offspring emerges

PP1-6 ectopic expression restores stumpy formation in RBP7 null mutants.

<p><b>A.</b> Inducible ectopic expression of PP1-6 in <i>T</i>. <i>brucei</i> EATRO 1125 AnTat1.1 90:13 parental cells (◼, +dox; ●, -dox) (*** p<0.0005). The dominant morphology of the cells on day 4 is shown schematically, which was stumpy in the induced samples and a mixture of slender, intermediate and stumpy in the uninduced samples. The inset northern blot shows PP1 transcript levels in the induced and uninduced cells; rRNA is the loading control. The right panel shows the expression of EP procyclin 4 hours after parasites were harvested on day 4 of infection and incubated in 6mM cis aconitate (*** p<0.0005). Infection terminated on humane grounds, ✝. <b>B.</b> Inducible ectopic expression of PP1-6 in <i>T</i>. <i>brucei</i> EATRO 1125 AnTat1.1 90:13 <i>RBP7AB</i> null mutants (◼, +dox; ●, -dox); (** p<0.005). The dominant morphology of the cells on day 4 is shown schematically. The inset northern blot shows PP1 transcript levels in the induced and uninduced cells; rRNA is the loading control. The right panel shows the expression of EP procyclin 4 hours after parasites were harvested on day 4 of infection and incubated in 6mM cis aconitate (* p<0.05). <b>C.</b> Morphology of <i>RBP7AB</i> null mutants induced (+dox) or not (-dox) to express PP1-6 (RBP7 KO PP1 OE). Bar = 10μm. <b>D.</b> Western blot of PAD1 expression in parental parasites (WT), RBP7AB null mutant (RBP7 KO) cells, or in parental cells (‘PP1 OE’) or in the null mutant (RBP7KO PP1 OE) induced (+dox) or not (-dox) to ectopically express PP1-6. The <i>RBP7AB</i> null mutant expresses PAD1 upon PP1-6 ectopic expression. EF1alpha provides a loading control.</p

NEK17 induced differentiation is dependent on RBP7.

<p><b>A.</b> Growth <i>in vivo</i> upon inducible ectopic expression of NEK17 in the <i>RBP7AB</i> null mutant. The dominant morphology of the cells on day 4 is shown schematically. Induced (+dox; ◼), uninduced (-dox; ●) over 4 days growth. <b>B.</b> Morphology of <i>RBP7AB</i> null mutants induced, or not, to ectopically express NEK17. Samples were isolated on day4 of infection. Bar = 10μm. <b>C.</b> Western blot demonstrating inducible ectopic expression of NEK17 in the <i>RBP7AB</i> null mutant. + dox, induced; -dox, uninduced. Elongation factor 1 alpha (EF1) provides the loading control. <b>D.</b> % 1K1N cells upon inducible ectopic expression of NEK17 in the <i>RBP7AB</i> null mutant at day 3 and day 4 post infection (* p<0.05; ** p<0.005). <b>E.</b> % PAD1 cells upon inducible ectopic expression of NEK17 in the <i>RBP7AB</i> null mutant at day 4 post infection (* p<0.05). Less PAD1 expression is detected in the NEK OE due to their lower overall parasitaemia than in the uninduced population (Fig 6A) with respect to RBP7AB KO cells alone (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007145#ppat.1007145.g002" target="_blank">Fig 2E</a>).</p

TbTOR4 drives growth arrest independently of MEKK1.

<p><b>A.</b> Parasitaemia of the <i>TbTOR4</i> RNAi line, <i>MEKK1</i> null mutant, or <i>TbTOR4</i> RNAi in the MEKK1 null mutant. In the left hand panel, <i>TbTOR4</i> silencing promotes stumpy formation; Doxycycline + (RNAi induced; ◼), doxycycline– (RNAi uninduced; ●). In the middle panel, the <i>MEKK1</i> null mutants (◼) are more virulent than parental (‘AnTat’) cells (●), consistent with the reduced capacity for stumpy formation. In the right-hand panel, when <i>TbTOR4</i> is depleted by inducible RNAi (◼) <i>MEKK1</i> null mutants show slow growth compared with <i>MEKK1</i> null mutants where <i>TbTOR4</i> is not depleted (●). Infections were humanely terminated in the uninduced <i>MEKK1</i> KO TbTOR4RNAi infections on day 4 and so day 5 data is absent (✝). *, p<0.05; **, p<0.005; ***, p<0.0005. <b>B.</b> Cell cycle progression of the cell lines analysed <i>in vivo</i> in Panel A. Left panel, <i>TbTOR4</i> RNAi shows enhanced levels of 1K1N cells at day 3 of infection, but by day 4 the uninduced cells have also progressed to stumpy forms. Middle panel, <i>MEKK1</i> null mutants show less accumulation in 1K1N than parental (‘AnTat’) cells. Right hand panel; when <i>TbTOR4</i> is depleted in the <i>MEKK1</i> null mutant, parasites show a higher proportion of 1K1N cells, such that TbTOR4 depletion overrides the absence of <i>MEKK1</i>. Infections were humanely terminated in the uninduced <i>MEKK1</i> KO <i>TbTOR4</i> RNAi infections on day 4 and so day 5 data is absent (✝). *, p<0.05; **, p<0.005; ***, p<0.0005. <b>C.</b> PAD1 expression when <i>TbTOR4</i> RNAi is induced in parental cells (left hand panel) or in the <i>MEKK1</i> null mutant (right hand panel). In the <i>TbTOR4</i> RNAi line on day 5 PAD1 expression in the uninduced cells exceeds the induced cells because these cells have progressed to stumpy at high parasitaemia; in contrast, the induced cells show incomplete <i>TbTOR4</i> RNAi and cells with effective knockdown express PAD1 early (day 3 and 4), while those without effective knockdown continue to proliferate as slender forms and express less PAD1 because the overall parasitaemia is lower. The middle panel shows less PAD1 is expressed in the absence of <i>MEKK1</i> compared with parental (‘AnTat’) cells. Infections were humanely terminated in the uninduced <i>MEKK1</i> KO TbTOR4 RNAi infections on day 4 and so day 5 data is absent (✝). *, p<0.05; **, p<0.005; ***, p<0.0005.</p

Phosphoproteomic analysis of a MEKK1 null mutant.

<p><b>A.</b> Experimental approach. <i>T</i>. <i>brucei</i> EATRO 1125 AnTat1.1 90:13 and <i>MEKK1</i> null mutants were grown in culture and harvested after identical growth profiles and at low cell density to maintain them as the slender developmental form. Samples were isolated, processed, subjected to isobaric mass tagging and then analysed by LC MS/MS. <b>B.</b> Reproducibility of the identified protein and phosphoprotein profiles derived from the replicates of the parental (‘AnTat1.1’) and <i>MEKK1</i> null mutant cells. <b>C.</b> Volcano plot analysis of phosphopeptide changes between the parental and <i>MEKK1</i> null mutant. Analyses focused on changes that were statistically significant (adjusted P value <0.05; shown with red dashed lines on the–log10 scale) and at least 1.5-fold different (shown as blue dashed lines on the Log2 scale) between the parental and <i>MEKK1</i> null mutant.</p