Intracellular proteome expression during 4-n-nonylphenol biodegradation by the filamentous fungus Metarhizium robertsii

4-n-nonylphenol (4-n-NP) is an endocrine disrupting compound (EDC); pollutants that cause serious disturbances in the environment. This study shows the degradation pathway and initial proteome analysis in cultures of a fungus that actively degrades 4-n-NP, Metarhizium robertsii. The research revealed the presence of 14 4-n-NP metabolites formed as a result of the oxidation of the alkyl chain and benzene ring, which leads to the complete decomposition of the compound. Based on the trend and quantitative analysis of the formation of 4-n-NP derivatives, the best conditions for proteome analysis were established. The data collected allowed the formulation of an explanation of the microorganism's strategy towards the removal of 4-n-NP. The main groups of proteins engaged in the removal of the xenobiotic are: oxidation-reduction systems related to nitroreductase-like proteins, ROS defense systems (peroxiredoxin and superoxide dismutase), the TCA cycle and energy-related systems. Principal components analysis was applied to unidentified proteins, resulting in the formulation of three subgroups and initial classification of these proteins

1,4-disubstituted thiosemicarbazide derivatives are potent inhibitors of toxoplasma gondii proliferation.

A series of 4-arylthiosemicarbazides substituted at the N1 position with a 5-membered heteroaryl ring was synthesized and evaluated in vitro for T. gondii inhibition proliferation and host cell cytotoxicity. At non-toxic concentrations for the host cells all studied compounds displayed excellent anti-parasitic effects when compared to sulfadiazine, indicating a high selectivity of their anti-T. gondii activity. The differences in bioactivity investigated by DFT calculations suggest that the inhibitory activity of 4-arylthiosemicarbazides towards T. gondii proliferation is connected with the electronic structure of the molecule. Further, these compounds were tested as potential antibacterial agents. No growth-inhibiting effect on any of the test microorganisms was observed for all the compounds, even at high concentrations

Toxoplasma gondii Recombinant antigen AMA1: Diagnostic Utility of Protein Fragments for the Detection of IgG and IgM Antibodies

Toxoplasma gondii is an important zoonotic protozoan that infects a wide variety of vertebrates as intermediate hosts. For this reason, the diagnosis of this disease is very important and requires continuous improvement. One possibility is to use recombinant antigens in serological tests. Apical membrane antigen 1 (AMA1), a protein located in specific secretory organelles (micronemes) of T. gondii, is very interesting in regard to its potential diagnostic utility. In the present study, we attempted to identify a fragment of the AMA1 protein with a high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. The full-length AMA1 and two different fragments (AMA1N and AMA1C) were produced using an Escherichia coli expression system. After purification by metal affinity chromatography, recombinant proteins were tested for their utility as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of IgG and IgM anti-T. gondii antibodies in human and mouse immune sera. Our data demonstrate that the full-length AMA1 recombinant antigen (corresponding to amino acid residues 67–569 of the native protein) has a better diagnostic potential than its N- or C-terminal fragments. This recombinant protein strongly interacts with specific anti-T. gondii IgG (99.4%) and IgM (80.0%) antibodies, and may be used for developing new tools for diagnostics of toxoplasmosis

Behavioral changes in mice caused by Toxoplasma gondii invasion of brain

Toxoplasma gondii, a protozoan parasite, is capable of infecting a broad range of intermediate warm-blooded hosts including humans. The parasite undergoes sexual reproduction resulting in genetic variability only in the intestine of the definitive host (a member of the cat family). The parasite seems to be capable of altering the natural behavior of the host to favor its transmission in the environment. The aim of this study was to evaluate the number of parasite cysts formed in the hippocampus and amygdala of experimentally infected mice as these regions are involved in defense behaviors control and emotion processing, and to assess the influence of the infection on mice behavior. The obtained results revealed the presence of parasite cysts both in the hippocampus and the amygdala of infected mice; however, no clear region-dependent distribution was observed. Furthermore, infected mice showed significantly diminished exploratory activity described by climbing and rearing, smaller preference for the central, more exposed part of the OF arena and engaged in less grooming behavior compared to uninfected controls

The Impact of the Antigenic Composition of Chimeric Proteins on Their Immunoprotective Activity against Chronic Toxoplasmosis in Mice

Toxoplasmosis may pose a serious threat for individuals with weakened or undeveloped immune systems. However, to date, there is no specific immunoprophylaxis for humans. Thus, the aim of this study was to evaluate the immunogenicity of three trivalent—SAG2-GRA1-ROP1L (SGR), SAG1L-MIC1-MAG1 (SMM), and GRA1-GRA2-GRA6 (GGG)—and two tetravalent—SAG2-GRA1-ROP1-GRA2 (SGRG) and SAG1-MIC1-MAG1-GRA2 (SMMG)—chimeric T. gondii proteins, as well as their protective potential against chronic toxoplasmosis in laboratory mice. All three trivalent recombinant proteins possessed immunogenic properties, as defined by specific humoral and cellular responses in vaccinated mice characterized by the synthesis of specific IgG (IgG1/IgG2a) antibodies in vivo and the release of Th1/Th2 cytokines by stimulated splenocytes in vitro. Immunization with all three recombinant proteins provided partial protection against toxoplasmosis, although the protective capacity strongly depended on the individual antigenic composition of each preparation. The antigens providing the highest (86%) and lowest (45%) protection, SGR and SMM, respectively, were supplemented with GRA2 antigen fragment, to form the tetravalent chimeric proteins SGRG and SMMG. Further study revealed that the tetravalent preparations exhibited high immunogenic potential; however, the addition of another antigen to the recombinant protein structure had distinct effects on the protection generated, compared to that of the trivalent counterparts, depending on the antigen tested

C5a-Preactivated Neutrophils Are Critical for Autoimmune-Induced Astrocyte Dysregulation in Neuromyelitis Optica Spectrum Disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune neuroinflammatory disease. In contrast to multiple sclerosis, autoantibodies against aquaporin-4 (AQP4) expressed on astrocytic end-feet have been exclusively detected in sera of NMOSD patients. Several lines of evidence suggested that anti-AQP4 autoantibodies are pathogenic, but the mechanism triggering inflammation, impairment of astrocyte function, and the role of neutrophils presented in NMOSD cerebrospinal fluid remains unknown. In this study, we tested how human neutrophils affect astrocytes in the presence of anti-AQP4 Ab-positive serum derived from NMOSD patients. An in vitro model of inflammation consisted of human astrocyte line, NMOSD serum, and allogenic peripheral blood neutrophils from healthy individuals. We showed evidence of pathogenicity of NMOSD serum, which by consecutive action of anti-AQP4 Abs, complement system, and neutrophils affected astrocyte function. Anti-AQP4 Ab binding astrocytes initiated two parallel complementary reactions. The first one was dependent on the complement cytotoxicity via C5b-9 complex formation, and the second one on the reverse of astrocyte glutamate pump into extracellular space by C5a-preactivated neutrophils. As a consequence, astrocytes were partially destroyed; however, a major population of astrocytes polarized into proinflammatory cells which were characterized by pathological glutamate removal from extracellular space

Phytoecdysteroids as modulators of the Toxoplasma gondii growth rate in human and mouse cells

Abstract Background Searching for new effective drugs against human and animal toxoplasmosis we decided to test the anti-Toxoplasma potential of phytoecdysteroids (α-ecdysone and 20-hydroxyecdysone) characterized by the pleiotropic activity on mammalian organisms including the enhancement of host’s anti-parasitic defence. This objective was accomplished by the in vitro evaluation of T. gondii growth in phytoecdysteroid-treated immunocompetent cells of selected hosts: humans and two strains of inbred mice with genetically determined different susceptibility to toxoplasmosis. Methods Peripheral mononuclear blood cells were isolated from Toxoplasma-positive and Toxoplasma-negative women (N = 43) and men (N = 21). Non-infected mice (C57BL/6, N = 10 and BALB/c, N = 14) and mice (BALB/c, N = 10) challenged intraperitoneally with 5 tissue cysts of the T. gondii DX strain were also used in this study as a source of splenocytes. The effects of phytoecdysteroids on the viability of human PBMC and mouse splenocytes were evaluated using the MTT assay. The influence of phytoecdysteroids on PBMCs, splenocytes and T. gondii proliferation was measured using radioactivity tests (the level of 3[H] uracil incorporation by toxoplasms or 3[H] thymidine by PBMCs and splenocytes), which was confirmed by quantitative Real-Time PCR. Statistical analysis was performed using SigmaStat 3.5 (Systat Software GmbH). The best-fit IC50 curves were plotted using GraphPad Prism 6.0 (GraphPad Software, Inc.). Results Our results showed that phytoecdysteroids promote the multiplication of Toxoplasma in cultures of human or murine immune cells, in contrast to another apicomplexan parasite, Babesia gibsoni. Additionally, the tested phytoecdysteroids did not stimulate the in vitro secretion of the essential protective cytokines (IFN-γ, IL-2 and IL-10), neither by human nor by murine immune cells involved in an effective intracellular killing of the parasite. Conclusions Judging by the effect of phytoecdysteroids on the T. gondii proliferation, demonstrated for the first time in this study, it seems that these compounds should not be taken into consideration as potential medications to treat toxoplasmosis. Phytoecdysteroids included in the food are most likely not harmful for human or animal health but certain nutrients containing ecdysteroids at high concentrations could promote T. gondii proliferation in chronically infected and immunocompromised individuals. In order to assess the real impact of ecdysteroids on the course of natural T. gondii invasion, in vivo research should be undertaken because it cannot be ruled out that the in vivo effect will be different than the in vitro one. However, taking into account the possible stimulating effect of ecdysteroids on some opportunistic parasites (such as Toxoplasma or Strongyloides) further studies are necessary and should focus on the mechanisms of their action, which directly or indirectly enhance the parasite growth. Since ecdysteroids are considered as potential drugs, it is essential to determine their effect on various parasitic pathogens, which may infect the host at the same time, especially in immunocompromised individuals

4-Arylthiosemicarbazide Derivatives as Toxoplasmic Aromatic Amino Acid Hydroxylase Inhibitors and Anti-inflammatory Agents

Approximately one-third of the human population is infected with the intracellular cosmopolitan protozoan Toxoplasma gondii (Tg), and a specific treatment for this parasite is still needed. Additionally, the increasing resistance of Tg to drugs has become a challenge for numerous research centers. The high selectivity of a compound toward the protozoan, along with low cytotoxicity toward the host cells, form the basis for further research, which aims at determining the molecular targets of the active compounds. Thiosemicarbazide derivatives are biologically active organic compounds. Previous studies on the initial preselection of 58 new 4-arylthiosemicarbazide derivatives in terms of their anti-Tg activity and selectivity made it possible to select two promising derivatives for further research. One of the important amino acids involved in the proliferation of Tg and the formation of parasitophorous vacuoles is tyrosine, which is converted by two unique aromatic amino acid hydroxylases to levodopa. Enzymatic studies with two derivatives (R: para-nitro and meta-iodo) and recombinant aromatic amino acid hydroxylase (AAHs) obtained in the E. coli expression system were performed, and the results indicated that toxoplasmic AAHs are a molecular target for 4-arylthiosemicarbazide derivatives. Moreover, the drug affinity responsive target stability assay also confirmed that the selected compounds bind to AAHs. Additionally, the anti-inflammatory activity of these derivatives was tested using THP1-Blue™ NF-κB reporter cells due to the similarity of the thiosemicarbazide scaffold to thiosemicarbazone, both of which are known NF-κB pathway inhibitors