Phosphorus deficiency induces sexual reproduction in the dinoflagellate Prorocentrum cordatum.

Nitrogen (N) and phosphorus (P) are essential elements whose availability promotes successful growth of phytoplankton and governs aquatic primary productivity. In this study, we investigated the effect of N and/or P deficiency on the sexual reproduction of Prorocentrum cordatum, the dinoflagellate with the haplontic life cycle which causes harmful algal blooms worldwide. In P. cordatum cultures, N and the combined N and P deficiency led to the arrest of the cell cycle in the G0/G1 phases and attenuation of cell culture growth. We observed, that P, but not N deficiency triggered the transition in the life cycle of P. cordatum from vegetative to the sexual stage. This resulted in a sharp increase in percentage of cells with relative nuclear DNA content 2C (zygotes) and the appearance of cells with relative nuclear DNA content 4C (dividing zygotes). Subsequent supplementation with phosphate stimulated meiosis and led to a noticeable increase in the 4C cell number (dividing zygotes). Additionally, we performed transcriptomic data analysis and identified putative phosphate transporters and enzymes involved in the phosphate uptake and regulation of its metabolism by P. cordatum. These include high- and low-affinity inorganic phosphate transporters, atypical alkaline phosphatase, purple acid phosphatases and SPX domain-containing proteins.

Zn2+ Intoxication of Mycobacterium marinum during Dictyostelium discoideum Infection Is Counteracted by Induction of the Pathogen Zn2+ Exporter CtpC.

Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum senses toxic levels of Zn2+ and responds by upregulating one of its isoforms of the Zn2+ efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn2+ as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR_1271 is the functional CtpC Zn2+ efflux transporter in M. marinum Infection leads to the accumulation of Zn2+ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn2+ efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn2+ into the MCV, carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn2+ than in wild-type cells, suggesting restriction by other Zn2+-mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn2+IMPORTANCE Microelements are essential for the function of the innate immune system. A deficiency in zinc or copper results in an increased susceptibility to bacterial infections. Zn2+ serves as an important catalytic and structural cofactor for a variety of enzymes including transcription factors and enzymes involved in cell signaling. But Zn2+ is toxic at high concentrations and represents a cell-autonomous immunity strategy that ensures killing of intracellular bacteria in a process called zinc poisoning. The cytosolic and lumenal Zn2+ concentrations result from the balance of import into the cytosol via ZIP influx transporters and efflux via ZnT transporters. Here, we show that Zn2+ poisoning is involved in restricting Mycobacterium marinum infections. Our study extends observations during Mycobacterium tuberculosis infection and explores for the first time how the interplay of ZnT transporters affects mycobacterial infection by impacting Zn2+ homeostasis.

Putative Meiotic Toolkit in the Dinoflagellate Prorocentrum cordatum: Additional Evidence for Sexual Process from Transcriptome.

Prorocentrum cordatum (Ostenfeld) Dodge-is a planktonic armored dinoflagellate that is a bloom-forming, potentially toxic cosmopolitan species. The transition from vegetative reproduction to the sexual process has been recently shown for this organism. Here, we present the results of transcriptomic data analysis that uncovered one syngamy-associated and 16 meiosis-associated proteins in P. cordatum. We also detected an amino acid sequence homologous to bacterial MutS2 protein. The MutS2 presence and origin in dinoflagellates are discussed for the first time.

Stressor-induced ecdysis and thecate cyst formation in the armoured dinoflagellates Prorocentrum cordatum.

Ecdysis, the process of extensive cell covering rearrangement, represents a remarkable physiological trait of dinoflagellates. It is involved in the regulation of the population and bloom dynamics of these microorganisms, since it is required for the formation of their thin-walled cysts. This study presents laboratory data on ecdysis in Prorocentrum cordatum, a harmful dinoflagellate species of high environmental significance. We studied external stressors triggering this process and changes in the cell ultrastructure accompanying it. Our experiments showed that mass ecdysis and formation of cysts in P. cordatum could be induced by centrifugation, temperature decrease, changes in salinity, and treatment by 2,6-dichlorobenzonitrile, whereas temperature increase, changes in pH and treatment by tetracycline did not have this effect. Obtained cysts of P. cordatum did not contain the pellicular layer and were formed in the end of the first stage of this process, i.e. removal of the plasma membrane and the outer amphiesmal vesicle membrane, whereas its second stage, removal of theca, represented excystment. Based on our findings, we conclude that such cysts can be attributed to thecate cysts and suggest P. cordatum as a promising model organism for the investigation of cellular and molecular aspects of ecdysis in dinoflagellates.

Life Cycle Stages and Evidence of Sexual Reproduction in the Marine Dinoflagellate Prorocentrum minimum (Dinophyceae, Prorocentrales).

Prorocentrum minimum is a potentially toxic marine dinoflagellate that often forms massive blooms in estuarine and coastal sea waters. In this study, the life cycle of P. minimum was investigated and sexual reproduction in culture was described for the first time. Morphology of the mitotic stages was revised and several distinguishing features from sexual steps were described. The sexual reproductive stages were observed in the stationary culture and compared with a well-studied closely related species, Prorocentrum micans. Prorocentrum minimum has a haplontic life cycle and homothallic sexual process. The gametes were isogamous and morphologically indistinguishable from the vegetative cells. Unlike P. micans, P. minimum isogametes fused, but did not conjugate, partially reorganizing their cell coverings. Newly formed planozygotes were distinguished by their irregular shape and a large asymmetrically located nucleus. No long-term resting cyst stages (hypnozygotes) were documented. The late planozygotes underwent meiosis and formed tetrads of cells. The second meiotic division could be delayed or arrested in one of the daughter nuclei leading to formation of trinucleate cells with three pairs of flagella. So, similar to P. micans, P. minimum may have two possible scenarios of sexual division: (a) formation of a four-cell stage through two successive divisions or (b) asynchronous divisions of the zygote. Changes in the DNA content were confirmed by quantitative image cytometry.

Localization of all four ZnT zinc transporters in Dictyostelium and impact of ZntA and ZntB knockout on bacteria killing.

Professional phagocytes have developed an extensive repertoire of autonomous immunity strategies to ensure killing of bacteria. Besides phagosome acidification and the generation of reactive oxygen species, deprivation of nutrients and the lumenal accumulation of toxic metals are essential to kill ingested bacteria or inhibit the growth of intracellular pathogens. Here, we used the soil amoeba Dictyostelium discoideum, a professional phagocyte that digests bacteria for nutritional purposes, to decipher the role of zinc poisoning during phagocytosis of nonpathogenic bacteria and visualize the temporal and spatial dynamics of compartmentalized, free zinc using fluorescent probes. Immediately after particle uptake, zinc is delivered to phagosomes by fusion with 'zincosomes' of endosomal origin, and also by the action of one or more zinc transporters. We localized the four Dictyostelium ZnT transporters to endosomes, the contractile vacuole and the Golgi complex, and studied the impact of znt knockouts on zinc homeostasis. We show that zinc is delivered into the lumen of Mycobacterium smegmatis-containing vacuoles, and that Escherichia coli deficient in the zinc efflux P1B-type ATPase ZntA are killed faster than wild-type bacteria.

STIM1 and STIM2 proteins differently regulate endogenous store-operated channels in HEK293 cells.

The endoplasmic reticulum calcium sensors stromal interaction molecules 1 and 2 (STIM1 and STIM2) are key modulators of store-operated calcium entry. Both these sensors play a major role in physiological functions in normal tissue and in pathology, but available data on native STIM2-regulated plasma membrane channels are scarce. Only a few studies have recorded STIM2-induced CRAC (calcium release-activated calcium) currents. On the other hand, many cell types display store-operated currents different from CRAC. The STIM1 protein regulates not only CRAC but also transient receptor potential canonical (TRPC) channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present for the first time experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. As shown in single-channel patch clamp experiments on HEK293 cells, selective activation of native STIM2 proteins or STIM2 overexpression results in store-operated activation of Imin channels, whereas STIM1 activation blocks this process. Changes in the ratio between active STIM2 and STIM1 proteins can switch the regulation of Imin channels between store-operated and store-independent modes. We have previously characterized electrophysiological properties of different Ca(2+) influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels; Imin channels are regulated by STIM2, TRPC3-containing INS channels are induced by STIM1, and TRPC1-composed Imax channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.