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      Acidification Activates Toxoplasma gondii Motility and Egress by Enhancing Protein Secretion and Cytolytic Activity

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      PLoS Pathogens
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          Abstract

          Pathogenic microbes rely on environmental cues to initiate key events during infection such as differentiation, motility, egress and invasion of cells or tissues. Earlier investigations showed that an acidic environment activates motility of the protozoan parasite T. gondii. Conversely, potassium ions, which are abundant in the intracellular milieu that bathes immotile replicating parasites, suppress motility. Since motility is required for efficient parasite cell invasion and egress we sought to better understand its regulation by environmental cues. We found that low pH stimulates motility by triggering Ca 2+-dependent secretion of apical micronemes, and that this cue is sufficient to overcome suppression by potassium ions and drive parasite motility, cell invasion and egress. We also discovered that acidification promotes membrane binding and cytolytic activity of perforin-like protein 1 (PLP1), a pore-forming protein required for efficient egress. Agents that neutralize pH reduce the efficiency of PLP1-dependent perforation of host membranes and compromise egress. Finally, although low pH stimulation of microneme secretion promotes cell invasion, it also causes PLP1-dependent damage to host cells, suggesting a mechanism by which neutral extracellular pH subdues PLP1 activity to allow cell invasion without overt damage to the target cell. These findings implicate acidification as a signal to activate microneme secretion and confine cytolytic activity to egress without compromising the viability of the next cell infected.

          Author Summary

          Toxoplasma and related parasites including those that cause malaria are obligate intracellular pathogens that replicate within a specialized compartment termed the parasitophorous vacuole. To infect new host cells these parasites must first escape from the parasitophorous vacuole and other limiting membranes of the currently infected cell. Escape, or egress as it is often called, depends on the timely release of adhesive proteins and lysis factors from secretory organelles called micronemes. Although this secretory event is crucial for egress, the natural environmental cues that trigger microneme secretion remain poorly defined. Here we discover that acidification of the parasitophorous vacuole is sufficient to trigger microneme secretion and promote the activity of a lysis factor called PLP1. We also show that pH-neutralizing drugs inhibit egress and provide evidence of parasitophorous vacuole acidification approximately coinciding with parasite egress from infected host cells. The findings support a working model in which acidification activates microneme dependent motility and lytic activity to execute egress and destruction of infected cells. The results also provide insight into how PLP1 lytic activity is stimulated during egress in an acidic environment and subsequently suppressed by the neutral extracellular environment, thus permitting cell invasion with minimal damage to the next target cell.

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          Most cited references27

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          Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito.

          Malaria is transmitted from vertebrate host to mosquito vector by mature sexual blood-living stages called gametocytes. Within seconds of ingestion into the mosquito bloodmeal, gametocytes undergo gametogenesis. Induction requires the simultaneous exposure to at least two stimuli in vitro: a drop in bloodmeal temperature to 5 degrees C below that of the vertebrate host, and a rise in pH from 7.4 to 8.0-8.2. In vivo the mosquito bloodmeal has a pH of between 7.5 and 7.6. It is thought that in vivo the second inducer is an unknown mosquito-derived gametocyte-activating factor. Here we show that this factor is xanthurenic acid. We also show that low concentrations of xanthurenic acid can act together with pH to induce gametogenesis in vitro. Structurally related compounds are at least ninefold less effective at inducing gametogenesis in vitro. In Drosophila mutants with lesions in the kynurenine pathway of tryptophan metabolism (of which xanthurenic acid is a side product), no alternative active compound was detected in crude insect homogenates. These data could form the basis of the rational development of new methods of interrupting the transmission of malaria using drugs or new refractory mosquito genotypes to block parasite gametogenesis.
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            Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma

            Calcium-regulated exocytosis is a ubiquitous process in eukaryotes, whereby secretory vesicles fuse with the plasma membrane and release their contents in response to an intracellular calcium surge1. This process regulates diverse cellular functions like plasma membrane repair in plants and animals2,3, discharge of defensive spikes in Paramecium 4, and secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons5. In animal cells, serine/threonine kinases including PKA, PKC and CaM-kinases have been implicated in calcium-signal transduction leading to regulated secretion1,6,7. Although plants and protozoa also regulate secretion via intracellular calcium, the means by which these signals are relayed have not been elucidated. Here we demonstrate that the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) is an essential regulator of calcium-dependent exocytosis in this opportunistic human pathogen. Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress. This phenotype was recapitulated using a chemical biology approach, wherein pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, since expression of a resistant kinase mutant reversed sensitivity to the inhibitor. TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates8, suggesting that related CDPKs may play a role in calcium-regulated secretion in other organisms. Since this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans.
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              Listeriolysin O: the Swiss army knife of Listeria.

              Listeriolysin O (LLO) is a toxin produced by Listeria monocytogenes, an opportunistic bacterial pathogen responsible for the disease listeriosis. This disease starts with the ingestion of contaminated foods and mainly affects immunocompromised individuals, newborns, and pregnant women. In the laboratory, L. monocytogenes is used as a model organism to study processes such as cell invasion, intracellular survival, and cell-to-cell spreading, as this Gram-positive bacterium has evolved elaborate molecular strategies to subvert host cell functions. LLO is a major virulence factor originally shown to be crucial for bacterial escape from the internalization vacuole after entry into cells. However, recent studies are revisiting the role of LLO during infection and are revealing new insights into the action of LLO, in particular before bacterial entry. These latest findings along with their impact on the infectious process will be discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                November 2014
                6 November 2014
                : 10
                : 11
                : e1004488
                Affiliations
                [1]Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
                University of Wisconsin Medical School, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MSR NS VBC. Performed the experiments: MSR NS. Analyzed the data: MSR NS VBC. Contributed reagents/materials/analysis tools: MSR NS. Wrote the paper: MSR VBC.

                [¤]

                Current address: Department of Pathology and Laboratory Medicine, Indiana University–Purdue University Indianapolis, Indianapolis, Indiana, United States of America

                Article
                PPATHOGENS-D-14-00940
                10.1371/journal.ppat.1004488
                4223073
                25375818
                0539ce91-2b19-496e-b9b9-7defead16ffe
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 April 2014
                : 24 September 2014
                Page count
                Pages: 11
                Funding
                MSR was supported by a University of Michigan Rackham Merit Fellowship, Rackham Student Research Grants, and by a Cellular and Molecular Biology Training Grant from the National Institutes of Health (T32 GM007315). This work was also supported by an operating grant from the National Institutes of Health Grant (R01AI046675 to VBC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Parasitology
                Medicine and Health Sciences
                Parasitic Diseases
                Protozoan Infections
                Toxoplasmosis
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. The data are available within the manuscript and the supporting information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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