A Structural and Functional Analysis of Toxoplasma gondii Perforin-like Protein 1.

Pore-forming proteins have been classically associated with host invasion from the bacterial perspective and cell lysis from the host perspective. However, we identified a new role for Toxoplasma gondii perforin-like protein 1 (PLP1) in rapid parasite exit from host cells (Kafsack et al., Science 2009). This places PLP1 at a unique junction combining vacuolar escape and cell lysis. I found that PLP1 is sufficient for membrane disruption and has a conserved mechanism of pore-formation. The highly conserved, central MACPF domain and the beta-sheet rich C-terminal domain were crucial for membrane permeabilization. Loss of the unique N-terminal extension reduced lytic activity and delayed egress, but did not significantly diminish virulence, suggesting that a small amount of lytic activity is still sufficient for pathogenesis. Both N- and C-terminal domains have membrane-binding activity, with the C-terminal domain being critical for function. This dual mode of membrane association may promote PLP1 activity and parasite egress in the diverse cell types in which this parasite replicates. Since the parasite must lyse the host cell upon egress and preserve intact host membranes upon invasion, I sought to identify mechanisms regulating lytic activity during egress and invasion. Using a hemolysis assay, I detected maximal PLP1 activity at low pH and minimal activity at neutral pH. Although the compartment in which the parasite replicates was presumed to be neutral pH, I found that pH-disrupting agents suppressed PLP1 activity during egress and reduced parasite exit. Modest decreases in vacuolar pH were observed when the parasite was exposed to egress cues and late in the replication cycle prior to native egress. Furthermore, low pH augments PLP1 activity during cell invasion, resulting in damage to host cells. These results support a model by which PLP1 activity is controlled by a pH switch: PLP1 is highly active in the low pH-environment during host cell exit, and is suppressed at neutral pH during host cell invasion. This work has uncovered novel structural and functional features of PLP1 pore formation. As parasite egress is a vulnerable point in the lytic cycle, further investigation of this process may identify potential targets for future therapeutics.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99994/1/mroiko_1.pd

New roles for perforins and proteases in apicomplexan egress

Egress is a pivotal step in the life cycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late-stage schizonts activate and secrete a subtilisin, PfSUB1, which processes enigmatic putative proteases called serine-repeat antigens that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75116/1/j.1462-5822.2009.01357.x.pd

Characterization of Pontibacter altruii, sp. nov., isolated from a human blood culture

The genus Pontibacter is a recent addition to the family Cytophagaceae, phylum Bacteroidetes. Previous reports of its cultivation and molecular detection are from a variety of environmental sources, including marine and desert habitats. We report the first description of a Pontibacter sp., which was initially identified as Elizabethkingia meningoseptica, isolated from a human clinical specimen. On the basis of 16S rRNA gene sequence, unique mass spectral profile and phenotypic characterization, this isolate represents a novel species within the genus Pontibacter that has been named Pontibacter altruii, sp. nov., strain Grand Forks

Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection

An unusual presentation of leishmaniasis in a human immunodeficiency virus-positive individual

INTRODUCTION: Leishmaniasis is a neglected tropical disease caused by vector-borne protozoa of the genus Leishmania. Cutaneous and mucocutaneous forms result in disfiguration or mutilation, whilst visceral leishmaniasis (VL) affects multiple organs and is fatal if untreated. Notably, Leishmania are capable of establishing a chronic infection, which may reactivate years after initial infection when the host becomes immune-suppressed. CASE PRESENTATION: A 24-year-old human immunodeficiency virus (HIV)-positive male presented for excision of anal condylomas. At the time of his current condyloma excision, the patient had no additional symptoms or cutaneous findings, but was noted to have been only intermittently compliant with his antiretroviral therapy. Microscopic examination of the haematoxylin and eosin-stained anal condyloma tissue revealed koilocytic change, ulceration and brisk histiocytic inflammation containing numerous small intracellular bodies suggestive of Leishmania amastigotes. A bone marrow biopsy was performed and demonstrated similar intracellular forms. Anal condyloma tissue and bone marrow aspirate were sent to the Centers for Disease Control and Prevention's Parasitic Diseases Branch for confirmation of Leishmania and speciation. Specific immunohistochemical staining for Leishmania in the tissue section was positive and the species was confirmed as Leishmania donovani by PCR. Subsequently, the patient resumed highly active antiretroviral therapy and received anti-Leishmania therapy. CONCLUSION: Whilst the presentation of VL in HIV-positive patients is often similar to those without HIV, here we describe an unusual initial presentation of leishmaniasis in an HIV-positive patient where the parasite was found in an anal condyloma. VL is a critical diagnosis that should be considered and pursued when leishmaniasis is encountered in seemingly illogical clinical settings

Acidification Activates <i>Toxoplasma gondii</i> Motility and Egress by Enhancing Protein Secretion and Cytolytic Activity

<div><p>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 <i>T. gondii</i>. 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 Ca²⁺-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.</p></div

Acidic pH increases invasion but results in host cell damage.

<p>(A) Low pH promotes parasite attachment and invasion. HFF cells were pulse invaded for 2 min with parasites in DMEM, or DMEM-like buffer at pH 5.4 or 7.4. *<i>p</i><0.05 vs. DMEM by student's <i>t</i>-test. Graph indicates the average and standard deviation of 3 independent experiments. (B) PLP1- and pH-dependent damage to host cells. HFF cells were preloaded with calcein-AM and WT and <i>plp1ko</i> parasites were allowed to settle on host cells in Endo buffer, which was subsequently switched to DMEM-like buffer of the indicated pH and incubated at 37°C for 10 min. Calcein signal in the supernatant was measured by fluorometry. Graph indicates average and standard deviation of triplicate wells and is representative of 3 independent experiments. *<i>p</i><0.05 vs. WT pH 7.4 by student's <i>t</i>-test.</p

Acidic pH induces parasite egress and pH shifts occur upon egress induction, and late in the replication cycle.

<p>(A) Parasite egress quantified by immunofluorescence microscopy. 30 h vacuoles were treated with high K⁺ buffer ±15 µM digitonin at 37°C for 3 min prior to fixation and egress enumeration. *<i>p</i><0.05 vs. pH 8.4 by student's <i>t</i>-test. Graph indicates the average and standard deviation of 3 independent experiments. (B) Superecliptic pHuorin signal is quenched at low pH. HFF cells were inoculated with increasing doses of superecliptic pHluorin expressing <i>plp1ko</i> parasites (<i>plp1ko</i>sepH) and fluorescence was observed 30 h post-infection in live cells or cells detergent lysed at pH 5.4 or 7.4. (C, D) A moderate decrease in pH occurs during induced egress. HFF inoculated with <i>plp1ko</i> parasites expressing superecliptic pHluorin for 30 h before treatment with DMSO or A23187 with or without 20 mM NH₄Cl (panel C) or 40 µM DCCD (panel D). Results indicate the average and standard deviation of triplicate wells and are representative of 3 independent experiments for NH₄Cl and 2 independent experiments for DCCD. * <i>p</i><0.05 vs. DMSO by student's <i>t</i>-test. (E) pH changes during parasite replication. Fluorescence signal without or with 20 mM NH₄Cl was followed for <i>plp1ko</i> expressing superecliptic pHluorin over the course of intracellular replication. The graph indicates the mean and standard deviation of triplicate wells and is representative of two independent experiments with independent clones. * <i>p</i><0.05 vs. 0 mM NH₄Cl by student's <i>t</i>-test.</p

pH neutralization suppresses parasite egress.

<p>(A–D) Parasite egress quantified by immunofluorescence microscopy. Wild type (RH) parasites were allowed to replicate for 30 h (A23187, Zaprinast) or 35 h (DTT) prior to pre-treatment with or without inhibitor followed by vehicle (DMSO, buffer) or egress inducer (2 µM A23187, 5 mM DTT, 250 µM Zaprinast) with or without inhibitor for 2 min. Immunofluorescence was performed for parasites (SAG1) and parasitophorous vacuole (GRA7) and occupied vacuoles were quantified. (*<i>p</i><0.05, student's <i>t</i>-test compared to A23187/DTT/Zaprinast alone). Graphs reflect the average and standard deviation of 3 independent experiment; * <i>p</i><0.05 by student's <i>t</i>-test vs. A23187/DTT/Zaprinast with no inhibitor.</p

Low pH promotes parasite motility and microneme secretion.

<p>(A) Videomicroscopy analysis of pH dependent parasite motility. Parasites were purified in high K⁺ buffer (pH 8.4) and analyzed before and after exchanging to the same buffer at pH 7.4 or 5.4. Graph indicates the average and standard deviation of 3 independent experiments. *<i>p</i><0.05 vs. pH 7.4 by student's <i>t</i>-test. (B) Immunoblot analysis of pH dependent microneme secretion. Parasites were purified in Endo buffer and switched to Endo buffer of the indicated pH, incubated for 2 min at 37°C and placed on ice. Blots of secreted material were probed with antibodies for MIC10, MIC4 or GRA1 as indicated. (C) Low pH induces more microneme secretion than ethanol stimulation and PLP1 is secreted and processed in a pH-dependent manner. Blots of secreted material were probed with antibodies for PLP1 and GRA4 as indicted. Also shown is a blot of PLP1 from cell pellets (cellular fraction) obtained by centrifugation after secretion. (D) Low-pH induced microneme secretion is blocked by the Ca²⁺ chelator, BAPTA-AM, as observed by immunoblot of PLP1, MIC2, MIC10, and MIC4. GRA1 secretion is not affected by BAPTA-AM. (E) Low pH induces microneme secretion in the presence of high K⁺ concentration. Arrowheads for panels B, D and E indicate products of SUB1 proteolysis.</p