scholarly journals Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Jillian E. Milanes ◽  
Jimmy Suryadi ◽  
Jan Abendroth ◽  
Wesley C. Van Voorhis ◽  
Kayleigh F. Barrett ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Homo Sapiens ◽  
Treatment Options ◽  
Amino Acid Identity ◽  
Pentose Phosphate ◽  
Mammalian Host ◽  
Binding Modes ◽  
Naegleria Fowleri ◽  
Content Type ◽  
Nægleria Fowleri

ABSTRACT Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (∼98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The first enzyme required for glucose metabolism is typically a hexokinase (HK), which transfers a phosphate from ATP to glucose. The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. However, the N. fowleri genome lacks an obvious HK homolog and instead harbors a glucokinase (Glck). The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (Homo sapiens Glck), suggesting that parasite-specific inhibitors with anti-amoeba activity can be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with the greatest activity observed with glucose. The enzyme had apparent Km values of 42.5 ± 7.3 μM and 141.6 ± 9.9 μM for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2-Å resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose-phosphorylating enzymes and identified several small molecules with 50% inhibitory concentration values of <1 μM that may prove useful as hit chemotypes for further leads and therapeutic development against N. fowleri.

scholarly journals Enzymatic and structural characterization of the Naegleria fowleri glucokinase

2018 ◽  
Author(s):  
Jillian E. Milanes ◽  
Jimmy Suryadi ◽  
Jan Abendroth ◽  
Wesley C. Van Voorhis ◽  
Kayleigh F. Barrett ◽  
...  
Keyword(s):  
Treatment Options ◽  
Structural Similarity ◽  
Amino Acid Identity ◽  
Pentose Phosphate ◽  
Mammalian Host ◽  
Binding Modes ◽  
Naegleria Fowleri ◽  
Therapeutic Development ◽  
Small Collection ◽  
Nægleria Fowleri

ABSTRACTInfection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited and the mortality rate is very high (~98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The genome of the parasite encodes a single glucose phosphorylating enzyme, a glucokinase (Glck). The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (HsGlck), suggesting that parasite-specific inhibitors with anti-amoeba activity could be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with greatest activity observed with glucose. The enzyme had apparent Km values of 42.5 ± 7.3 μM and 141.6 ± 9.9 μM for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2 Å resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose phosphorylating enzymes and identified several small molecules with IC50 values < 1 μM that may prove useful as hit chemotypes for further lead and therapeutic development against N. fowleri.

scholarly journals Naegleria fowleri: Pathogenesis, Diagnosis, and Treatment Options

2015 ◽  
Vol 59 (11) ◽  
pp. 6677-6681 ◽  
Author(s):  
Eddie Grace ◽  
Scott Asbill ◽  
Kris Virga
Keyword(s):  
Case Reports ◽  
Treatment Options ◽  
Clinical Manifestations ◽  
Naegleria Fowleri ◽  
Content Type ◽  
High Profile ◽  
Free Living Amoeba ◽  
Nægleria Fowleri ◽  
Amoebic Meningoencephalitis

ABSTRACTNaegleria fowlerihas generated tremendous media attention over the last 5 years due to several high-profile cases. Several of these cases were followed very closely by the general public.N. fowleriis a eukaryotic, free-living amoeba belonging to the phylum Percolozoa.Naegleriaamoebae are ubiquitous in the environment, being found in soil and bodies of freshwater, and feed on bacteria found in those locations. WhileN. fowleriinfection appears to be quite rare compared to other diseases, the clinical manifestations of primary amoebic meningoencephalitis are devastating and nearly always fatal. Due to the rarity ofN. fowleriinfections in humans, there are no clinical trials to date that assess the efficacy of one treatment regimen over another. Most of the information regarding medication efficacy is based on either case reports orin vitrostudies. This review will discuss the pathogenesis, diagnosis, pharmacotherapy, and prevention ofN. fowleriinfections in humans, including a brief review of all survivor cases in North America.

scholarly journals Vaccination with Lentiviral Vector Expressing thenfa1Gene Confers a Protective Immune Response to Mice Infected with Naegleria fowleri

2013 ◽  
Vol 20 (7) ◽  
pp. 1055-1060 ◽  
Author(s):  
Jong-Hyun Kim ◽  
Hae-Jin Sohn ◽  
Jinyoung Lee ◽  
Hee-Jong Yang ◽  
Yong-Joon Chwae ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune Responses ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Survival Rates ◽  
Dna Vaccination ◽  
Naegleria Fowleri ◽  
Content Type ◽  
Ifn Γ ◽  
Nægleria Fowleri

ABSTRACTNaegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. Thenfa1gene (360 bp), cloned from a cDNA library ofN. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. Thenfa1gene plays an important role in the pathogenesis ofN. fowleriinfection. To examine the effect ofnfa1DNA vaccination againstN. fowleriinfection, we constructed a lentiviral vector (pCDH) expressing thenfa1gene. For thein vivomouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing thenfa1gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing thenfa1gene also exhibited significantly higher survival rates (90%) after challenge withN. fowleritrophozoites. Finally, thenfa1vaccination effectively induced protective immunity by humoral and cellular immune responses inN. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool againstN. fowleriinfection.

scholarly journals Comparative genome sequence analysis of several species in the genus Tepidimonas and the description of a novel species Tepidimonas charontis sp. nov.

2020 ◽  
Vol 70 (3) ◽  
pp. 1596-1604 ◽  
Author(s):  
Luciana Albuquerque ◽  
Nadine Castelhano ◽  
Pedro Raposo ◽  
Hugo J. C. Froufe ◽  
Igor Tiago ◽  
...  
Keyword(s):  
Type Species ◽  
Hot Spring ◽  
Novel Species ◽  
Amino Acid Identity ◽  
Pentose Phosphate ◽  
Rrna Gene ◽  
Content Type ◽  
Optimum Growth Temperature ◽  
Link Type ◽  
Conserved Genes

We performed high-quality genome sequencing of eight strains of the species of the genus Tepidimonas and examined the genomes of closely related strains from the databases to understand why Tepidimonas taiwanensis is the only strain of this genus that utilizes glucose and fructose for growth. We found that the assimilation of these hexoses by T. taiwanensis was due to the presence of two transporters that are absent in all other genomes of strains of members of the genus Tepidimonas examined. Some strains lack genes coding for glucokinase, but the Embden–Meyerhof–Parnas pathway appears to be otherwise complete. The pentose phosphate pathway has a complete set of genes, but genes of the Entner–Doudoroff pathway were not identified in the genomes of any of the strains examined. Genome analysis using average nucleotide identity (ANIb), digital DNA–DNA hybridization (dDDH), average amino acid identity (AAI) and phylogenetic analysis of 400 conserved genes was performed to assess the taxonomic classification of the organisms. Two isolates of the genus Tepidimonas from the hot spring at São Pedro do Sul, Portugal, designated SPSP-6T and SPSPC-18 were also examined in this study. These organisms are mixotrophic, have an optimum growth temperature of about 50 ºC, utilize several organic acids and amino acids for growth but do not grow on sugars. Distinctive phenotypic, 16S rRNA gene sequence and genomic characteristics of strains SPSP-6T and SPSPC-18 lead us to propose a novel species based on strain SPSP-6T for which we recommend the name Tepidimonas charontis sp. nov. (=CECT 9683T=LMG 30884T).

Neuroinfecção por Naegleria fowleri:

1999 ◽  
Vol 15 (4) ◽  
Author(s):  
Rodrigo Siqueira Batista ◽  
Andréia Patrícia Gomes ◽  
David B Oddó ◽  
Luiz Eduardo de Oliveira Viana ◽  
Renata Cristina Teixeira Pinto ◽  
...  
Keyword(s):  
Homo Sapiens ◽  
Naegleria Fowleri ◽  
Nægleria Fowleri

As amebas de vida livre são seres vivos que se distribuem amplamente no planeta e, em determinadas circunstâncias, podem provocar infecções em animais, dentre os quais o Homo sapiens sapiens. Neste âmbito, vem merecendo destaque a espécie Naegleria fowleri, capaz de desencadear quadros extremamente graves, habitualmente de difícil abordagem diagnóstica e terapêutica, o que culmina, no mais das vezes, em alta letalidade. Com base nessas premissas, revisar os principais aspectos da infecção humana por N. fowleri é escopo do presente artigo.

Naegleria fowleri: diagnosis, treatment options and pathogenesis

2019 ◽  
Vol 7 (2) ◽  
pp. 67-80 ◽  
Author(s):  
Mohammad Ridwane Mungroo ◽  
Naveed Ahmed Khan ◽  
Ruqaiyyah Siddiqui
Keyword(s):  
Treatment Options ◽  
Naegleria Fowleri ◽  
Nægleria Fowleri

scholarly journals Counting Legionella Cells within Single Amoeba Host Cells

2012 ◽  
Vol 78 (6) ◽  
pp. 2070-2072 ◽  
Author(s):  
Helen Y. Buse ◽  
Nicholas J. Ashbolt
Keyword(s):  
Legionella Pneumophila ◽  
Water Systems ◽  
Host Cells ◽  
Naegleria Fowleri ◽  
Acanthamoeba Polyphaga ◽  
Content Type ◽  
Cell Numbers ◽  
Nægleria Fowleri

ABSTRACTHere we present the first attempt to quantifyLegionella pneumophilacell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturableL. pneumophilacells grown withinAcanthamoeba polyphagaandNaegleria fowleriwere 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite−1, respectively.

scholarly journals NLRP3 Inflammasome Activation in THP-1 Target Cells Triggered by Pathogenic Naegleria fowleri

2016 ◽  
Vol 84 (9) ◽  
pp. 2422-2428 ◽  
Author(s):  
Jong-Hyun Kim ◽  
Hae-Jin Sohn ◽  
Jong-Kyun Yoo ◽  
Heekyoung Kang ◽  
Gi-Sang Seong ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Macrophage ◽  
Target Cells ◽  
Inflammasome Activation ◽  
Naegleria Fowleri ◽  
Content Type ◽  
Inflammatory Reactions ◽  
Caspase 1 ◽  
Nægleria Fowleri

Naegleria fowleri, known as the brain-eating amoeba, causes acute primary amoebic meningoencephalitis. During swimming and other recreational water activities,N. fowleritrophozoites penetrate the nasal mucosa and invade the olfactory bulbs, resulting in intense inflammatory reactions in the forebrain tissue. To investigate what kinds of inflammasome molecules are expressed in target cells due toN. fowleriinfection, human macrophage cells (THP-1 cells) were cocultured withN. fowleritrophozoites in a noncontact system, and consequently, interleukin-1β (IL-1β) production was estimated. Caspase-1 activation and IL-1β production from THP-1 cells by Western blotting and the culture supernatant by enzyme-linked immunosorbent assay analysis were observed at 3 h after cocultivation. In addition, the increased expression of ASC and NLRP3, which make up an inflammasome complex, was also observed at 3 h after cocultivation. To confirm the caspase-1 activation and IL-1β production via the NLRP3 inflammasome in THP-1 cells triggered byN. fowleritrophozoites, THP-1 cells were pretreated with several inhibitors. The inhibition assay showed that CA-074 (a cathepsin B inhibitor), glybenclamide (an NLRP3 molecule inhibitor), andN-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) reduced the levels of caspase-1 activation and IL-1β production from THP-1 cells. This study suggests thatN. fowleriinfection induces the NLRP3 inflammasome, which activates caspase-1 and subsequently produces IL-1β, thus resulting in inflammation.

scholarly journals Bis-Benzimidazole Hits against Naegleria fowleri Discovered with New High-Throughput Screens

2015 ◽  
Vol 59 (4) ◽  
pp. 2037-2044 ◽  
Author(s):  
Christopher A. Rice ◽  
Beatrice L. Colon ◽  
Mehmet Alp ◽  
Hakan Göker ◽  
David W. Boykin ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
High Throughput ◽  
Lead Optimization ◽  
New Drugs ◽  
Naegleria Fowleri ◽  
Onset Of Action ◽  
Content Type ◽  
Nægleria Fowleri

ABSTRACTNaegleria fowleriis a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two newin vitroassays suitable for medium- or high-throughput drug discovery and used these forN. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono- and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC50) of 177 nM. Similarly, we identified 10 additional analogues with IC50s of <1 μM, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono- and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections withN. fowleri.

Hypothermia: depression of tricarboxylic acid cycle flux and evidence for pentose phosphate shunt upregulation

1999 ◽  
Vol 90 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Taro Kaibara ◽  
Garnette R. Sutherland ◽  
Fred Colbourne ◽  
Randy L. Tyson
Keyword(s):  
Steady State ◽  
Glucose Metabolism ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Pentose Phosphate ◽  
Water Soluble ◽  
Resonance Spectroscopy ◽  
Acid Extraction ◽  
Content Type ◽  
Fine Print

Object. Hypothermia is used in neurosurgery and other surgical disciplines to reduce tissue injury, but the mechanism of such protection remains elusive. The authors have endeavored to delineate the mechanism of neural protection afforded by hypothermia through a study of glucose metabolism.Methods. Nuclear magnetic resonance spectroscopy was used to follow the carbon-13 label from [1-13C]glucose as it was metabolized through the glycolytic and tricarboxylic acid pathways. Male Sprague—Dawley rats were maintained at either 37.5°C or 31°C and infused with labeled glucose for 10, 30, 60, 100, or 200 minutes (five rats were used for each time point and for each temperature). At the end of the infusion period, the rats' brains were subjected to rapid freeze-funnel fixation. Water-soluble metabolites were extracted from samples of the neocortex and hippocampus by using perchloric acid extraction. The fractional enrichment of these metabolites was used to calculate the reaction rate constant of formation and steady-state enrichment for a number of metabolites.Hypothermia resulted in a 30 to 40% depression of metabolism (p < 0.0001) in both the neocortex and hippocampus. Steady-state fractional enrichment of metabolites was also decreased by 20 to 25% with hypothermia (p < 0.0001), implying a loss of label during metabolism.Conclusions. The results of this study suggest that an increased fraction of glucose metabolism was shunted through the pentose phosphate pathway in the presence of hypothermia.

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