The greenbeard gene tgrB1 regulates altruism and cheating in Dictyostelium discoideum.

Greenbeard genetic elements encode rare perceptible signals, signal recognition ability, and altruism towards others that display the same signal. Putative greenbeards have been described in various organisms but direct evidence for all the properties in one system is scarce. The tgrB1-tgrC1 allorecognition system of Dictyostelium discoideum encodes two polymorphic membrane proteins which protect cells from chimerism-associated perils. During development, TgrC1 functions as a ligand-signal and TgrB1 as its receptor, but evidence for altruism has been indirect. Here, we show that mixing wild-type and activated tgrB1 cells increases wild-type spore production and relegates the mutants to the altruistic stalk, whereas mixing wild-type and tgrB1-null cells increases mutant spore production and wild-type stalk production. The tgrB1-null cells cheat only on partners that carry the same tgrC1-allotype. Therefore, TgrB1 activation confers altruism whereas TgrB1 inactivation causes allotype-specific cheating, supporting the greenbeard concept and providing insight into the relationship between allorecognition, altruism, and exploitation.

The polyketide synthase StlA is involved in inducing aggregation in Polysphondylium violaceum.

In the social amoeba Dictyostelium discoideum, the polyketide MPBD (4-methyl-5-pentylbenzene-1,3-diol) regulates the gene expressions of cAMP signaling to make cells aggregation-competent and also induces spore maturation. The polyketide synthase StlA is responsible for MPBD biosynthesis in D. discoideum and appears to be conserved throughout the major groups of the social amoeba (Dictyostelia). In this study, we analyzed the function of StlA in Polysphondylium violaceum by identifying the gene sequence and creating the knockout mutants. We found that Pv-stlA- mutants had defects only in cell aggregation but not in spore maturation, indicating that the function of StlA in inducing spore maturation is species-specific. We also found that MPBD could rescue the aggregation defect in Pv-stlA- mutants whereas the mutants normally exhibited chemotaxis to their chemoattractant, glorin. Our data suggest that StlA is involved in inducing aggregation in P. violaceum by acting on signaling pathways other than chemotaxis in P. violaceum.

CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.

Role of a Concentration Gradient in Malaria Drug Resistance Evolution: A Combined within- and between-Hosts Modelling Approach.

Resistance to antimalarial drugs is currently a growing public health problem, resulting in more cases with treatment failure. Although previous studies suggested that a concentration gradient facilitates the antibiotic resistance evolution in bacteria, no attempt has been made to investigate the roles of a concentration gradient in malaria drug resistance. Unlike the person-to-person mode of transmission of bacteria, the malaria parasites need to switch back and forth between the human and mosquito hosts to complete the life cycle and to spread the resistant alleles. Here we developed a stochastic combined within- and between-hosts evolutionary dynamics model specific to malaria parasites in order to investigate the influence of an antimalarial concentration gradient on the evolutionary dynamics of malaria drug resistance. Every stage of malaria development in both human and mosquito hosts are individually modelled using the tau-leaping algorithm. We found that the concentration gradient can accelerate antimalarial resistance evolution. The gain in resistance evolution was improved by the increase in the parasite mutation rate and the mosquito biting rate. In addition, even though the rate of resistance evolution is not sensitive to the changes in parasite reduction ratios (PRRs) of antimalarial drugs, the probability of finding the antimalarial drug resistant parasites decreases when the PRR increases.

Native parasite affecting an introduced host in aquaculture: cardiac henneguyosis in the red seabream Pagrus major Temminck & Schlegel (Perciformes: Sparidae) caused by Henneguya aegea n. sp. (Myxosporea: Myxobolidae).

BACKGROUND: Henneguya Thélohan, 1892 (Myxobolidae) is one of the most species-rich genera of myxosporean parasites infecting fish. Although common in nature, there are few reports of these parasites causing important disease in aquaculture. In this paper, we describe a new species of Henneguya infecting Pagrus major (Temminck & Schlegel), a fish host introduced to the Mediterranean Sea from Japan in the late 1980s. RESULTS: Large plasmodia of the parasite were found in the bulbus arteriosus and in the ventricle of the infected fish. Spores were found mainly in the kidney and heart and were accompanied by melanized macrophages or vascular intimal proliferation mixed with a mild non-suppurative response, respectively. Comparisons of morphometric data for spore and polar capsule length and width, suggest a unique combination of features in the newly described species. Molecular analysis, based on 18S rDNA sequence of the parasite, followed by phylogenetic analysis, indicated that the parasite described here is a novel species of Henneguya, clustered with the marine congeneric species. CONCLUSIONS: Henneguya aegea n. sp. infects in aquaculture P. major, a host introduced as eggs to the Mediterranean from Japan. Despite the high host specificity of the myxobolid parasites, H. aegea n. sp. seems to be able to use P. major as a host and propagate successfully, causing morbidity and mortality. This could result in spillback of the new species from high density cultured non-native P. major to native fish hosts.

Real-Time PCR and LAMP Assays for the Detection of Spores of Alternaria solani and Sporangia of Phytophthora infestans to Inform Disease Risk Forecasting.

Real-time loop-mediated isothermal amplification (LAMP) assays for the detection of sporangia of the causal pathogen of late blight, Phytophthora infestans, and spores of the main causal pathogen of early blight, Alternaria solani, were developed to facilitate the in-field detection of airborne inoculum to improve disease forecasting. These assays were compared with an existing real-time PCR assay for P. infestans and a newly developed real-time PCR assay for A. solani. Primers were designed for real-time LAMP of P. infestans and A. solani. The specificity of the P. infestans real-time LAMP assay was similar to that of an existing real-time PCR assay: DNA of P. infestans was consistently amplified as was DNA of the taxonomically closely related species Phytophthora mirabilis, Phytophthora phaseoli, and Phytophthora ipomoea; no amplification of DNA from the potato pathogens Phytophthora erythroseptica or Phytophthora nicotianae occurred. Real-time LAMP and PCR assays were developed for A. solani, and the specificity was compared with an existing conventional PCR assay. Importantly, the A. solani real-time LAMP and PCR assays did not amplify the species Alternaria alternata. However, cross-reactivity with Alternaria dauci was observed with the real-time PCR assay and Alternaria brassicae with the real-time LAMP assay. The sensitivity of all assays for the detection of DNA extracted from sporangia/spores of the target pathogens was evaluated. The P. infestans real-time LAMP assay reliably detected 5 pg of DNA, equivalent to ∼1 sporangia per reaction. By comparison, 20 fg of DNA was detectable with the existing real-time PCR assay. In the case of A. solani, real-time LAMP detected 4.4 pg of DNA, equivalent to ∼1 spore per reaction, and real-time PCR detected 200 fg of DNA. In-field air samplers were deployed in two trial plots planted with potato: one infected with P. infestans, and the other infected with A. solani. Four additional samplers were located in commercial potato fields. Air samples were taken through the season, and detection of airborne inoculum of P. infestans and A. solani with both real-time PCR and LAMP was assessed.

Phylogeny of Myxobolidae (Myxozoa) and the evolution of myxospore appendages in the Myxobolus clade.

Genera Myxobolus Bütschli, 1882 and Henneguya Thélohan, 1892 (Myxobolidae) are specious myxozoan genera. They comprise nearly half of overall known myxozoan species diversity. A typical spore feature of Henneguya is the presence of two caudal appendages of the spore valves, which distinguishes them from species of the genus Myxobolus. Several Myxobolus spp., however, were reported to show aberrant spores with Henneguya-like caudal appendages. We found such aberrant spores in Myxobolus tsangwuensis and Myxobolus wulii. We studied the ultrastructure of M. wulii and Myxobolus oralis spores with caudal appendages by transmission electron microscopy (TEM). TEM of these aberrant spores revealed that their caudal appendages have the same ultrastructure as the appendages of Henneguya spp. Small caudal appendages of M. wulii spores observed only on TEM suggested that this character may be often overlooked and more Myxobolus species potentially have the ability to express the caudal appendages on the myxospore. In order to trace the evolution of this character, we performed broad phylogenetic analysis of all species of the family Myxobolidae which are available in GenBank including nearly 300 taxa. We found at least eight independent evolutionary origins of spores with two appendages, three origins of a single appendage and 12 apparent secondary losses of the spore projections. Therefore, genus Henneguya with typical two-tailed myxospores is polyphyletic, however a majority of its species has a common ancestor and groups in the second largest subclade of the Myxobolus clade. We also mapped the biological characteristics (host, site of infection and environment) of Myxobolidae species on the phylogenetic tree. We revealed an evident host-associated evolutionary pattern in all parts of the Myxobolus clade with a distinct and species-rich subclade containing almost exclusively species infecting species of the Order Cypriniformes.

First Myxozoan Infection (Cnidaria: Myxosporea) in a Marine Polychaete from North America and Erection of Actinospore Collective Group Saccimyxon.

In a survey of marine annelids for myxosporean infection in Charleston Harbor, South Carolina, we collected 3,214 polychaetes from 21 families and found infections in 6 spionid individuals. Based on gross morphology and COI sequencing, all infected spionids were identified as Streblospio benedicti. Infection prevalence was 0.8% (6/734) of that species of spionid, and 0.2% of all 3,214 polychaetes examined. Pansporocysts contained 8 actinospores and developed in the tegument of the annelid host. This is the first myxozoan infection recorded from this polychaete species, second in the family, and the first marine myxozoan found in the Americas. It is the first marine species found to develop in the tegument of its annelid host; a site of development observed only once before, in Ceratonova shasta infections of its freshwater sabellid polychaete host. Mature actinospores were morphologically simple, truncated ellipsoids, lacking processes or ornamentation, 9.0 ± 0.5 µm × 6.0 ± 0.4 µm. Their sack-like shape was similar to 9 of the 12 actinospores described previously from polychaetes; 10/12 had been and ascribed originally to the morphological collective group Tetractinomyxon despite 9 of these having few similarities to the original description of this group. We propose to name the simple, spherical to ellipsoidal spore morphotype Saccimyxon to encompass both our novel actinospore and the 9 other sack-like polychaete actinospore types in the literature. In the present study, 18S rDNA sequencing demonstrated that the myxozoans that infected the 6 spionids were genetically the same species (type sample 1,737 nucleotides, GenBank accession number MH791159) and was not >95% similar to any sequence in GenBank. Phylogenetic analysis showed that the myxozoan species we encountered is basal to the kudoids and thus likely to have a morphologically simple myxospore stage with fewer than 4 valves. However, without a genetic match, the presumptive vertebrate host remains unknown.

4-Methyl-5-Pentylbenzene-1,3-Diol Regulates Chemotactic Cell Aggregation and Spore Maturation Via Different Mechanisms in Dictyostelium discoideum.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of the polyketide synthase SteelyA, is a signaling molecule that regulates Dictyostelium discoideum development. During early development, MPBD controls chemotactic cell aggregation by regulating the expression of genes in the cAMP signaling pathway; however, during culmination at late development, it induces spore maturation. In the present study, we analyzed the effects of MPBD, its derivatives, and a putative MPBD-derived metabolite on developmental defects in the MPBD-less stlA null mutant. Using structure-activity relationship studies, it was observed that in MPBD, the functional groups that were essential for induction of spore maturation were different from those essential for induction of cell aggregation. Dictyoquinone, a putative MPBD metabolite rescued the aggregation defect in stlA null mutant in early development, but not the spore maturation defect at the later stage. Our data suggest that MPBD regulates chemotactic cell aggregation and spore maturation via different mechanisms.

Proto-oncogenes in a eukaryotic unicellular organism play essential roles in plasmodial growth in host cells.

BACKGROUND: The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. RESULTS: Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. CONCLUSIONS: Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

A MADS-box transcription factor regulates a central step in sporulation of the oomycete Phytophthora infestans.

MADS-box transcription factors play significant roles in eukaryotes, but have not yet been characterized in oomycetes. Here, we describe a MADS-box protein from Phytophthora infestans, which causes late blight of potato. P. infestans and most other oomycetes express a single MADS-box gene. PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillate in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing triggered by a construct expressing 478 nt of MADS sequences indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes.

Cellular Events of Multinucleated Giant Cells Formation During the Encystation of Entamoeba invadens.

Entamoeba histolytica, the causative agent of amoebiasis, does not form cysts in vitro, so reptilian pathogen Entamoeba invadens is used as an Entamoeba encystation model. During the in vitro encystation of E. invadens, a few multinucleated giant cells (MGC) were also appeared in the culture along with cysts. Like the cyst, these MGC's were also formed in the multicellular aggregates found in the encystation culture. Time-lapse live cell imaging revealed that MGC's were the result of repeated cellular fusion with fusion-competent trophozoites as a starting point. The early MGC were non-adherent, and they moved slowly and randomly in the media, but under confinement, MGC became highly motile and directionally persistent. The increased motility resulted in rapid cytoplasmic fissions, which indicated the possibility of continuous cell fusion and division taking place inside the compact multicellular aggregates. Following cell fusion, each nucleus obtained from the fusion-competent trophozoites gave rise to four nuclei with half genomic content. All the haploid nuclei in MGC later aggregated and fused to form a polyploid nucleus. These observations have important implications on Entamoeba biology as they point toward the possibility of E. invadens undergoing sexual or parasexual reproduction.

Alkaline treatment of resting spores prior to DNA extraction improves the purity of Plasmodiophora brassicae DNA.

A commonly used protocol for DNA extraction from Plasmodiophora brassicae was modified by adding an alkaline treatment step to increase the purity of resting spores. The quality of DNA extracted by the modified protocol was improved due to the removal of DNA contamination from host plant cells and other microorganisms.

The transcription factor Spores Absent A is a PKA dependent inducer of Dictyostelium sporulation.

Sporulation in Dictyostelium fruiting bodies evolved from amoebozoan encystation with both being induced by cAMP acting on PKA, but with downstream components still being unknown. Using tagged mutagenesis to find missing pathway components, we identified a sporeless mutant defective in a nuclear protein, SpaA. Expression of prespore genes was strongly reduced in spaA- cells, while expression of many spore stage genes was absent. Chromatin immunoprecipitation (ChIP) of a SpaA-YFP gene fusion showed that (pre)spore gene promoters bind directly to SpaA, identifying SpaA as a transcriptional regulator. SpaA dependent spore gene expression required PKA in vivo and was stimulated in vitro by the membrane-permeant PKA agonist 8Br-cAMP. The PKA agonist also promoted SpaA binding to (pre)spore promoters, placing SpaA downstream of PKA. Sequencing of SpaA-YFP ChIPed DNA fragments revealed that SpaA binds at least 117 (pre)spore promoters, including those of other transcription factors that activate some spore genes. These factors are not in turn required for spaA expression, identifying SpaA as the major trancriptional inducer of sporulation.

Encystation: the most prevalent and underinvestigated differentiation pathway of eukaryotes.

Not long ago, protists were considered one of four eukaryote kingdoms, but recent gene-based phylogenies show that they contribute to all nine eukaryote subdomains. The former kingdoms of animals, plants and fungi are now relegated to lower ranks within subdomains. Most unicellular protists respond to adverse conditions by differentiating into dormant walled cysts. As cysts, they survive long periods of starvation, drought and other environmental threats, only to re-emerge when conditions improve. For protists pathogens, the resilience of their cysts can prevent successful treatment or eradication of the disease. In this context, effort has been directed towards understanding the molecular mechanisms that control encystation. We here firstly summarize the prevalence of encystation across protists and next focus on Amoebozoa, where most of the health-related issues occur. We review current data on processes and genes involved in encystation of the obligate parasite Entamoeba histolytica and the opportunistic pathogen Acanthamoeba. We show how the cAMP-mediated signalling pathway that controls spore and stalk cell encapsulation in Dictyostelium fruiting bodies could be retraced to a stress-induced pathway controlling encystation in solitary Amoebozoa. We highlight the conservation and prevalence of cAMP signalling genes in Amoebozoan genomes and the suprisingly large and varied repertoire of proteins for sensing and processing environmental signals in individual species.

An individual-level selection model for the apparent altruism exhibited by cellular slime moulds.

In Dictyostelium discoideum, cells that become part of the stalk or basal disc display behaviour that can be interpreted as altruistic. Atzmony et al. (Curr Sci 72:142-145, 1997) had hypothesised that this behaviour could be the outcome of an adaptive strategy based on differing intrinsic quality as reflected by phenotypes that indicate differences in potential for survival and reproduction, followed by intercellular competition among amoebae of differing qualities. Low-quality amoebae would have a poor chance of succeeding in the competition to form spores; they could enhance their chances of survival by adopting a presumptive stalk strategy. Here we extend the hypothesis by making use of recent findings. Our approach is based on the view that an evolutionary explanation for the apparent altruism of stalk cells in D. discoideum must apply broadly to other cellular slime moulds (CSMs) that exhibit stalk cell death. Further, it must be capable of being modified to cover social behaviour in CSMs with an extracellular stalk, as well as in sorocarpic amoebae whose stalk cells are viable. With regard to D. discoideum, we suggest that (a) differentiation-inducing factor, thought of as a signal that inhibits amoebae from forming spores and induces them to differentiate into basal disc cells, is better viewed as a mediator of competition among post-aggregation amoebae and (b) the products of the 'recognition genes', tgrB and tgrC, allow an amoeba to assess its quality relative to that of its neighbours and move to a position within the aggregate that optimises its reproductive fitness. From this perspective, all cells behave in a manner that is 'selfish' rather than 'altruistic', albeit with different expectations of success.

The immunophilin repertoire of Plasmodiophora brassicae and functional analysis of PbCYP3 cyclophilin.

Plasmodiophora brassicae is a soil-borne pathogen that belongs to Rhizaria, an almost unexplored eukaryotic organism group. This pathogen requires a living host for growth and multiplication, which makes molecular analysis further complicated. To broaden our understanding of a plasmodiophorid such as P. brassicae, we here chose to study immunophilins, a group of proteins known to have various cellular functions, including involvement in plant defense and pathogen virulence. Searches in the P. brassicae genome resulted in 20 putative immunophilins comprising of 11 cyclophilins (CYPs), 7 FK506-binding proteins (FKBPs) and 2 parvulin-like proteins. RNAseq data showed that immunophilins were differentially regulated in enriched life stages such as germinating spores, maturing spores, and plasmodia, and infected Brassica hosts (B. rapa, B. napus and B. oleracea). PbCYP3 was highly induced in all studied life stages and during infection of all three Brassica hosts, and hence was selected for further analysis. PbCYP3 was heterologously expressed in Magnaporthe oryzae gene-inactivated ΔCyp1 strain. The new strain ΔCyp1+ overexpressing PbCYP3 showed increased virulence on rice compared to the ΔCyp1 strain. These results suggest that the predicted immunophilins and particularly PbCYP3 are activated during plant infection. M. oryzae is a well-studied fungal pathogen and could be a valuable tool for future functional studies of P. brassicae genes, particularly elucidating their role during various infection phases.

Characterization and expression analysis of a new small heat shock protein Hsp20.4 from Eimeria tenella.

Small heat shock proteins (sHsps) are ubiquitous and diverse molecular chaperones. Found in almost all organisms, they regulate protein refolding and protect cells from stress. Until now, no sHsp has been characterized in Eimeria tenella. In this study, the novel EtsHsp20.4 gene was cloned from E. tenella by rapid amplification of cDNA ends based on a previously identified expressed sequence tag. The full-length cDNA was 1019bp in length and contained an open reading frame of 558bp that encoded a 185-amino acid polypeptide with a calculated molecular weight of 20.4 kDa. The EtsHsp20.4 protein contained a distinct HSP20/alpha-crystallin domain that is the key determinant of their function as molecular chaperones and belongs to the HSP20 protein family. EtsHsp20.4 mRNA levels were higher in sporulated oocysts than in sporozoites or second-generation merozoites by real-time quantitative PCR, the transcription of EtsHsp20.4 was barely detectable in unsporulated oocysts. Immunolocalization with EtsHsp20.4 antibody showed that EtsHsp20.4 was mainly located on the surface of sporozoites, first-generation merozoites and second-generation merozoites. Following the development of parasites in DF-1 cells, EtsHsp20.4 protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts. Malate dehydrogenase thermal aggregation assays indicated that recombinant EtsHsp20.4 had molecular chaperone activity in vitro. These results suggested that EtsHsp20.4 might be involved in sporulation in external environments and intracellular growth of the parasite in the host.

Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer.

Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca- structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP-induced cAMP synthesis as well as c-di-GMP-induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca- mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer.

Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion.

UNLABELLED: Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia's sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-Rac(CA)) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-Rac(CA)-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis. IMPORTANCE: The encystation process is crucial for the transmission of giardiasis and the life cycle of many protists. Encystation for Giardia lamblia involves the assembly of a protective cyst wall via sequential production, trafficking, and secretion of cyst wall material. However, the regulatory pathways that coordinate cargo maturation and secretion remain unknown. Here, we asked whether the signaling activities of G. lamblia's single Rho family GTPase, GlRac, might have a regulatory role in the encystation process. We show that GlRac localizes to endomembranes and its signaling activities regulate the production of cyst wall protein 1 (CWP1), the maturation of encystation-specific vesicles (ESVs), and secretion of CWP1. We also show that secreted CWP1 is available for the development of cysts at the population level, a finding that in part could explain why Giardia encystation proceeds more efficiently at high cell densities.