Toxoplasma gondii Actively Inhibits Neuronal Function in Chronically Infected Mice

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii–infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca2+) imaging studies revealed that tachyzoites actively manipulated Ca2+ signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca2+ uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca2+ stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host

LEKTI domain 15 is a functional Kazal-type proteinase inhibitor

The multidomain proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor) consists of 15 potential serine proteinase inhibitory domains. In various diseases such as the severe skin disorder Netherton syndrome as well as atopy, defects in the gene encoding LEKTI have been identified that generate premature termination codons of translation, suggesting a specific role of the COOH-terminal part of LEKTI in healthy individuals. We overexpressed and purified a sequence comprising the 15th domain of LEKTI for further characterisation. Here, we present a high yield expression system for recombinant production and efficient purification of LEKTI domain 15 as a highly soluble protein with a uniform disulfide pattern that is identical to that of other known Kazal-type inhibitors. Also, the expected P1P1′ site was confirmed. LEKTI domain 15 is a well-structured protein as verified by circular dichroism (CD) spectroscopy and a tight-binding and stable inhibitor of the serine proteinase trypsin. These findings confirm the designation of domain 15 as a proteinase inhibitor of the Kazal family

Domain 15 of the serine proteinase inhibitor LEKTI blocks HIV infection <em>in vitro</em>

<p><strong>Background:</strong> Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a 15-domain serine proteinase inhibitor, parts of which have first been isolated from human blood filtrate. It is encoded by the gene SPINK5. In the past, different groups reported antiviral activities of certain serine proteinase inhibitors, such as mucous proteinase inhibitor and alpha1-proteinase inhibitor. The purpose of this study was to test two representative domains of the proteinase inhibitor LEKTI for anti-HIV activities.</p><p><strong>Methods:</strong> LEKTI domains 6 and 15 were recombinantly produced in <em>E.coli</em>. To test their inhibitory activity against HIV infection, the reporter cell line P4-R5 MAGI carrying an HIV-inducible reporter gene was infected by a CCR5-tropic HIV strain in the presence of different inhibitor concentrations. After three days, infection rates were determined by quantifying ß-galactosidase activities using the Galacto-Light Plus™ ß-Galactosidase Reporter Gene Assay.</p><p><strong>Results:</strong> In contrast to LEKTI domain 6, LEKTI domain 15 suppressed HIV-induced reporter gene activities with an IC₅₀ value of approximately 29 µM.</p><p><strong>Conclusion:</strong> LEKTI domain 15 represents an inhibitor of HIV infection. <em><strong>(Med J Indones. 2013;22:131-5. doi: 10.13181/mji.v22i3.580)</strong></em></p><p><strong>Keywords:</strong> <em>HIV, inhibition, LEKTI, P4-R5 MAGI</em></p

The number of intracerebral T. gondii cysts declines over time in the total brain but not in brain regions important for behaviour.

<p>The total number of <i>T. gondii</i> cysts was evaluated on complete <i>T. gondii</i>-immunostained brain section series at day 30 (n = 4) and 60 p.i. (n = 5). (<i>A</i>) The number significantly declined from day 30 to 60 p.i. (p<0.05). (<i>B</i>) However, the number of <i>T. gondii</i> cysts significantly declined only in the indicated (*) brain regions (p<0.05) but not in the remaining brain regions.</p

Disturbed Ca²⁺ response in glutamate-stimulated, tachyzoite infected cortical neurons in vitro.

<p>(<i>A–C</i>) Tachyzoites infect dendrites (<i>A</i>), soma (<i>B</i>), and axons (<i>C</i>) of cultivated cortical neurons as revealed by double immunofluorescence anti-<i>T. gondii</i> (red fluorescent) and anti-class III β-tubulin staining (green fluorescent). <i>(D)</i> Live cell Ca2⁺ imaging of non-infected control and tachyzoite-infected neurons upon stimulation with glutamate. The time course of Ca2⁺ response is shown as three serial images at 0, 500, and 1000 seconds. Red arrow exhibits a hyper-responsive infected cell while the white arrows demonstrate hypo-responsive infected cells. A counter immunofluorescence staining of the infected culture was done with anti-<i>T. gondii</i> showing the presence of toxoplasma tachyzoits in the imaged neurons. <i>(E)</i> A graphical representation of calcium responses elicited by controls and infected neurons; control (n = 6), infected hyper-responsive (n = 7), infected hypo-responsive (n = 7). Data are represented as mean ± SEM. (<i>F</i>) Neurons infected with heat killed <i>T. gondii</i> in-vitro showed no changes in calcium responses upon glutamate stimulation. (<i>G</i>) Thapsigargin induces a huge calcium response in the uninfected controls (n = 8) which does not regress due to the inability to reuptake calcium in the endoplasmic stores, but the infected neurons (n = 9) show only a weak signalling upon thapsigargin-stimulation indicating a depletion of intracellular calcium stores.</p