Reassessment of the Role of Aromatic Amino Acid Hydroxylases and the Effect of Infection by Toxoplasma gondii on Host Dopamine

Toxoplasma gondiiinfection has been described previously to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylaseAAH2gene, with no observable phenotype in parasite growth or differentiationin vitroandin vivo. Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine eitherin vitroorin vivo, even whenAAH2was overexpressed using theBAG1promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may affect this pathway locally. Additionally, our findings suggest alternative roles for theAHHenzymes inT. gondii, sinceAAH1is essential for growth in nondopaminergic cells.

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Role of Toxoplasma gondii Chloroquine Resistance Transporter in Bradyzoite Viability and Digestive Vacuole Maintenance

mBio ◽

10.1128/mbio.01324-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 7

Author(s):

Geetha Kannan ◽

Manlio Di Cristina ◽

Aric J. Schultz ◽

My-Hang Huynh ◽

Fengrong Wang ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Chronic Infection ◽

Chloroquine Resistance ◽

Congenital Defects ◽

Functional Link ◽

Content Type ◽

Chloroquine Resistance Transporter

ABSTRACT Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow-growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosome-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum chloroquine resistance transporter (CRT), TgCRT, has previously been identified in T. gondii. To interrogate the function of TgCRT in chronic-stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and the viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo. Interestingly, impairing VAC proteolysis in TgCRT-deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This study provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection. IMPORTANCE Individuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well-validated potential targets. We show here that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasite’s lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.

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Genetic Basis forIn VitroandIn VivoResistance to Lincosamides, Streptogramins A, and Pleuromutilins (LSAP Phenotype) in Enterococcus faecium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01030-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4463-4469 ◽

Cited By ~ 28

Author(s):

Christophe Isnard ◽

Brigitte Malbruny ◽

Roland Leclercq ◽

Vincent Cattoir

Keyword(s):

Amino Acid ◽

Molecular Mechanism ◽

Enterococcus Faecium ◽

Full Genome Sequence ◽

Comparative Genomic ◽

Content Type ◽

Abc Protein

ABSTRACTAs opposed toEnterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A),Enterococcus faeciumis naturally susceptible. Since this phenotype may be selected forin vivoby quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance inE. faecium. Six LSAP-resistantin vitromutants ofE. faeciumHM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of anin vitromutant (E. faeciumUCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in allin vitroandin vivoresistant strains but was not present in susceptible strains. The wild-type allele was namedeat(A) (forEnterococcusABCtransporter), and its mutated allelic variant was namedeat(A)v. The introduction ofeat(A)vfrom UCN90B into HM1070 conferred the LSAP phenotype, whereas that ofeat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance inE. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.

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Bradyzoite Pseudokinase 1 Is Crucial for Efficient Oral Infectivity of the Toxoplasma gondii Tissue Cyst

Eukaryotic Cell ◽

10.1128/ec.00343-12 ◽

2013 ◽

Vol 12 (3) ◽

pp. 399-410 ◽

Cited By ~ 35

Author(s):

Kerry R. Buchholz ◽

Paul W. Bowyer ◽

John C. Boothroyd

Keyword(s):

Toxoplasma Gondii ◽

Acid Treatment ◽

Cyst Formation ◽

Oral Infection ◽

Tissue Cyst ◽

Content Type ◽

Oral Transmission

ABSTRACTThe tissue cyst formed by the bradyzoite stage ofToxoplasma gondiiis essential for persistent infection of the host and oral transmission. Bradyzoite pseudokinase 1 (BPK1) is a component of the cyst wall, but nothing has previously been known about its function. Here, we show that immunoprecipitation of BPK1 fromin vitrobradyzoite cultures, 4 days postinfection, identifies at least four associating proteins: MAG1, MCP4, GRA8, and GRA9. To determine the role of BPK1, a strain ofToxoplasmawas generated with thebpk1locus deleted. This BPK1 knockout strain (Δbpk1) was investigatedin vitroandin vivo. No defect was found in terms ofin vitrocyst formation and no difference in pathogenesis or cyst burden 4 weeks postinfection (wpi) was detected after intraperitoneal (i.p.) infection withΔbpk1tachyzoites, although the Δbpk1cysts were significantly smaller than parental or BPK1-complemented strains at 8 wpi. Pepsin-acid treatment of 4 wpiin vivocysts revealed that Δbpk1parasites are significantly more sensitive to this treatment than the parental and complemented strains. Consistent with this, 4 wpi Δbpk1cysts showed reduced ability to cause oral infection compared to the parental and complemented strains. Together, these data reveal that BPK1 plays a crucial role in thein vivodevelopment and infectivity ofToxoplasmacysts.

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“In vivo” amplification of biological activity of tetragastrin by amino acid hydroxamates

Bioscience Reports ◽

10.1007/bf01116693 ◽

1984 ◽

Vol 4 (12) ◽

pp. 1009-1015 ◽

Cited By ~ 2

Author(s):

J. P. Bali ◽

H. Mattras ◽

A. Previero ◽

M. A. Coletti-Previero

Keyword(s):

Biological Activity ◽

Amino Acid ◽

Aromatic Amino Acid ◽

Aminopeptidase Activity ◽

Proteolytic Degradation ◽

Short Peptides ◽

Rat Blood ◽

Active Peptides

Rat blood was shown to contain an aminopeptidase which rapidly hydrolyses short peptides containing an aromatic amino acid as N-terminal residue. Using tetragastrin (Trp-Met-Asp-PheNH 2) as substrate, we showed that some amino acid hydroxamates inhibit rat aminopeptidase activity ‘in vitro’ in the following order: HTrpNHOH > HPheNHOH ≫ HAIaNHOH. The same hydroxamates markedly enhanced the biological activity of tetragastrin ‘in vivo’. The amplification of the secretory effect, correlated with the amount of the hydroxamate used, strongly suggests that these compounds can stabilize a number of active peptides in vivo by inhibiting their proteolytic degradation.

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Antagonists of the system L neutral amino acid transporter (LAT) promote endothelial adhesivity of human red blood cells

Thrombosis and Haemostasis ◽

10.1160/th16-05-0373 ◽

2017 ◽

Vol 117 (07) ◽

pp. 1402-1411 ◽

Cited By ~ 5

Author(s):

Laura Beth Mann Dosier ◽

Vikram J. Premkumar ◽

Hongmei Zhu ◽

Izzet Akosman ◽

Michael F. Wempe ◽

...

Keyword(s):

Amino Acid ◽

Red Blood Cells ◽

Blood Cells ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Neutral Amino Acid Transporter ◽

System L

SummaryThe system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.

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Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

Infection and Immunity ◽

10.1128/iai.00959-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 15

Author(s):

Jonathan L. Portman ◽

Qiongying Huang ◽

Michelle L. Reniere ◽

Anthony T. Iavarone ◽

Daniel A. Portnoy

Keyword(s):

Protein Function ◽

Inhibitory Effect ◽

Cysteine Residue ◽

Infection Model ◽

Listeriolysin O ◽

Content Type ◽

Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

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Fimbria-Encoding GeneyadCHas a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

Infection and Immunity ◽

10.1128/iai.01138-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 187-193 ◽

Cited By ~ 12

Author(s):

Renu Verma ◽

Thaís Cabrera Galvão Rojas ◽

Renato Pariz Maluta ◽

Janaína Luisa Leite ◽

Livia Pilatti Mendes da Silva ◽

...

Keyword(s):

Escherichia Coli ◽

Soft Agar ◽

Pleiotropic Effect ◽

Biological Characteristics ◽

Wild Type ◽

Content Type ◽

Pathogenic Escherichia Coli

The extraintestinal pathogen termed avian pathogenicEscherichia coli(APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07.In vitro, the transcription level ofyadCwas upregulated at 41°C and downregulated at 22°C. TheyadCexpressionin vivowas more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadCstrain presented a slightly decreased ability to adhere to HeLa cells with or without thed-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed thatfimHwas downregulated (P< 0.05) andcsgAandecpAwere slightly upregulated in the mutant strain, showing thatyadCmodulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadCstrain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P< 0.05). Motility assays in soft agar demonstrated that the ΔyadCstrain was less motile than the wild type (P< 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadCstrain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

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The functional dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels

Biochemical Journal ◽

10.1042/bj20030115 ◽

2004 ◽

Vol 377 (1) ◽

pp. 25-36 ◽

Cited By ~ 50

Author(s):

Stéphanie MOUHAT ◽

Amor MOSBAH ◽

Violeta VISAN ◽

Heike WULFF ◽

Muriel DELEPIERRE ◽

...

Keyword(s):

Amino Acid ◽

Scorpion Toxin ◽

Xenopus Laevis Oocytes ◽

Amino Acid Residues ◽

Voltage Gated ◽

Toxin Binding ◽

Ic50 Values

Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 µg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 µM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

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Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

Clinical and Vaccine Immunology ◽

10.1128/cvi.00351-12 ◽

2012 ◽

Vol 19 (10) ◽

pp. 1603-1608 ◽

Cited By ~ 20

Author(s):

Koushik Roy ◽

David J. Hamilton ◽

James M. Fleckenstein

Keyword(s):

Escherichia Coli ◽

Diarrheal Disease ◽

Vaccine Development ◽

Enterotoxigenic Escherichia Coli ◽

Intestinal Colonization ◽

Content Type ◽

Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

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