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  1. Majid MI, Akmal DH, Few LL, Agustien A, Toh MS, Samian MR, et al.
    Int J Biol Macromol, 1999 Jun-Jul;25(1-3):95-104.
    PMID: 10416655
    A locally isolated soil microorganism identified as Erwinia sp. USMI-20 has been found to produce poly(3-hydroxybutyrate), P(3HB), from either palm oil or glucose and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), from a combination of palm oil and a second carbon source of either one of the following compounds: propionic acid, n-propanol, valeric acid and n-pentanol. It was found that Erwinia sp. USMI-20 could produce P(3HB) up to 69 wt.% polymer content with a dry cell weight of 4.4 g/l from an initial amount of 14.5 g/l of glucose followed by a feeding rate of glucose at 0.48 g/h glucose. On the other hand, the bacteria can achieve 46 wt.% of P(3HB) and a dry cell weight of 3.6 g/l from a batch fermentation in a 10-l fermentor from an initial concentration of 4.6 g/l of palm oil. Further characterisation of the polymer production was also carried out by using different types of palm oil. Among the different palm oils that were used, crude palm oil was the best lipid source for P(3HB) production as compared to palm olein and palm kernel oil. In the production of the copolymer, P(3HB-co-3HV), the highest mole fraction of 3-HV units could be as high as 47 mol% from a single feeding of valeric acid upon initial growth on palm oil.
  2. Too WC, Liew YC, Few LL
    J Basic Microbiol, 2008 Oct;48(5):430-5.
    PMID: 18759222 DOI: 10.1002/jobm.200800008
    Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterium codenamed pi9 was selected for the cloning of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway. Here, the cloning of an 1,113 bp fragment of GAPDH gene which covers the 1,002 bp open reading frame by using multiple PCR steps is described. The partial sequence of this gene was PCR amplified by using degenerate primers followed by the cloning of the flanking sequences by inverse and splinkerette PCR techniques. The success in cloning the GAPDH gene by PCR has bypassed the more time consuming genomic library construction and screening method. The full length GAPDH protein was subsequently expressed in E. coli, purified as His-tag protein and confirmed to be catalytically active. This work demonstrated the use of multiple PCR techniques to clone a gene based solely on sequence comparison. It also laid the foundation for further biochemical and structural characterizations of GAPDH from a psychrophilic bacterium by providing a highly purified recombinant protein sample.
  3. See Too WC, Few LL
    World J Microbiol Biotechnol, 2010 Jul;26(7):1251-9.
    PMID: 24026930 DOI: 10.1007/s11274-009-0295-9
    Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterial isolate codenamed π9 was selected for the cloning of the gene encoding triose phosphate isomerase (TIM), an enzyme in the glycolytic pathway. Based on 16S rRNA gene sequence analysis, this isolate was identified as a species of the genus Pseudomonas under the P. fluorescens group. The cloning of a 816 bp fragment of TIM gene which covers the 756 bp open reading frame was achieved by a combination of degenerate and splinkerette PCRs. The partial sequence of this gene was first PCR amplified by using degenerate primers and the flanking sequences were subsequently amplified by splinkerette PCR technique. Amino acid sequence of the cloned TIM was 97% identical to TIM from Pseudomonas fluorescens and shared 51% identity with the TIM from psychrophilic Vibrio sp. This work demonstrated the use of multiple PCR techniques to clone a gene without prior knowledge of its sequence. The cloning of the TIM gene by PCR was more rapid and cost effective compared to the traditional genomic library construction and screening method. Homology model of the TIM protein in this study was generated based on Escherichia coli TIM crystal structure. The model could serve as a hypothetical TIM structure from a psychrophilic microorganism for further investigation into areas that showed deviations from the known mesophilic TIM structures.
  4. Too WC, Wong MT, Few LL, Konrad M
    PLoS One, 2010;5(9):e12999.
    PMID: 20886003 DOI: 10.1371/journal.pone.0012999
    Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.
  5. Chua SL, See Too WC, Khoo BY, Few LL
    Cytotechnology, 2011 Dec;63(6):645-54.
    PMID: 21850463 DOI: 10.1007/s10616-011-9383-4
    Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C(t) values. The geNorm analysis showed the following ranking for stability of genes: UBC, YWHAZ > RPLP > TBP > ACTB > HPRT1 > PPIA > GAPDH > GUSB > B2M > TUBB > RRN18S. A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.
  6. Kuan CS, Wong MT, Choi SB, Chang CC, Yee YH, Wahab HA, et al.
    Int J Mol Sci, 2011;12(7):4441-55.
    PMID: 21845088 DOI: 10.3390/ijms12074441
    Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.
  7. Wong MT, Choi SB, Kuan CS, Chua SL, Chang CH, Normi YM, et al.
    Int J Mol Sci, 2012;13(1):901-17.
    PMID: 22312293 DOI: 10.3390/ijms13010901
    Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity.
  8. Kuan CS, Yee YH, See Too WC, Few LL
    PLoS One, 2014;9(12):e113485.
    PMID: 25490397 DOI: 10.1371/journal.pone.0113485
    Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.
  9. Fong SW, Few LL, See Too WC, Khoo BY, Nik Ibrahim NN, Yahaya SA, et al.
    BMC Res Notes, 2015;8:679.
    PMID: 26576922 DOI: 10.1186/s13104-015-1677-8
    Biomarkers play a pivotal role in the diagnosis and management of patients with acute coronary syndrome. This study aimed to investigate the differences in level of several biomarkers, i.e. C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor, between acute coronary syndrome and chronic stable angina patients. The relationship between these biomarkers in the coronary circulation and systemic circulation was also investigated.
  10. Chang CC, Few LL, Konrad M, See Too WC
    PLoS One, 2016;11(5):e0154702.
    PMID: 27149373 DOI: 10.1371/journal.pone.0154702
    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.
  11. Kuan CS, See Too WC, Few LL
    PLoS One, 2016;11(1):e0147886.
    PMID: 26807725 DOI: 10.1371/journal.pone.0147886
    Ethanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied.
  12. Wong WK, Foo PC, Olivos-Garcia A, Noordin R, Mohamed Z, Othman N, et al.
    Acta Trop, 2017 Aug;172:208-212.
    PMID: 28506795 DOI: 10.1016/j.actatropica.2017.05.017
    Crude soluble antigen (CSA) produced from Entamoeba histolytica trophozoite is conventionally used for serodiagnosis of invasive amoebiasis. However, high background seropositivities by CSA-assay in endemic areas complicate the interpretation of positive result in clinical settings. Instead, incorporating a second assay which indicates active or recent infection into the routine amoebic serology could possibly complement the limitations of CSA-assay. Hence, the present study aimed to evaluate the diagnostic efficacies of indirect ELISAs using CSA and excretory-secretory antigen (ESA) for serodiagnosis of amoebic liver abscess (ALA). Reference standard for diagnosis of ALA at Hospital Universiti Sains Malaysia is based on clinical presentation, radiological imaging and positive indirect haemagglutination assay (titer ≥256). Five groups of human serum samples collected from the hospital included Group I - ALA diagnosed by the reference standard and pus aspirate analysis using real-time PCR (n=10), Group II - ALA diagnosed by the reference standard only (n=41), Group III - healthy control (n=45), Group IV - other diseases control (n=51) and Group V - other infectious diseases control (n=31). For serodiagnosis of ALA serum samples (Group I and II), CSA-ELISA showed sensitivities of 100% for both groups, while ESA-ELISA showed sensitivities of 100% and 88%, respectively. For serodiagnosis of non-ALA serum samples (Group III, IV and V), CSA-ELISA showed specificities of 91%, 75% and 100%, respectively; while ESA-ELISA showed specificities of 96%, 98% and 100%, respectively. Indirect ELISAs using CSA and ESA have shown distinct strength for serodiagnosis of ALA, in terms of sensitivity and specificity, respectively. In conclusion, parallel analysis by both assays improved the overall efficacies of amoebic serology as compared to either single assay.
  13. Ayub Khan SM, Few LL, See Too WC
    Mol Med Rep, 2018 May;17(5):7442-7450.
    PMID: 29568919 DOI: 10.3892/mmr.2018.8762
    Choline kinase (CK) is the first enzyme in the CDP-choline pathway for the synthesis of phosphatidylcholine, the most abundant phospholipid in the mammalian cell membrane. This enzyme exists as three isozymes (α1, α2 and β) and the CKα isozyme has been implicated in cancer pathogenesis. Inhibition of CK activity has been proposed for cancer therapies. MicroRNAs (miRNAs/miRs) are non‑coding RNAs that serve important roles in diverse biological pathways and human diseases, including cancer. However, the regulation of CKα gene expression by miRNAs has never been investigated, to the best of the authors' knowledge. In the present study, two miRNA mimics, miR‑876‑5p and miR‑646, were transfected into the HepG2 cell line and the effect of these miRNAs on the levels of CKα mRNA were determined by reverse transcription‑quantitative polymerase chain reaction. Cells transfected with 25 nM miR‑876‑5p for 48 h exhibited significantly lower levels of CKα mRNA. Following optimization, miR‑876‑5p caused four times lower levels of CKα mRNA compared to the negative control. Effects of the miRNAs on HepG2 cell viability and cellular morphology were additionally analyzed using an MTT cell viability assay and scanning electron microscopy, respectively. HepG2 cells that were transfected with the optimum concentration of miR‑876‑5p for the optimum duration exhibited 25% lower viability than negative control and signs of apoptosis in electron micrographs. The results suggested miR‑876‑5p as a potential miRNA modulator of CKα expression in the cells, and may be relevant for the design of more effective anticancer strategy targeting CK.
  14. Khalifa M, Few LL, See Too WC
    Biomed Res Int, 2020;2020:1823485.
    PMID: 32695809 DOI: 10.1155/2020/1823485
    Novel antimicrobial agents are crucial to combat antibiotic resistance in pathogenic bacteria. Choline kinase (ChoK) in bacteria catalyzes the synthesis of phosphorylcholine, which is subsequently incorporated into the cell wall or outer membrane. In certain species of bacteria, phosphorylcholine is also used to synthesize membrane phosphatidylcholine. Numerous human ChoK inhibitors (ChoKIs) have been synthesized and tested for anticancer properties. Inhibition of S. pneumoniae ChoK by human ChoKIs showed a promising effect by distorting the cell wall and retarded the growth of this pathogen. Comparison of amino acid sequences at the catalytic sites of putative choline kinases from pathogenic bacteria and human enzymes revealed striking sequence conservation that supports the potential application of currently available ChoKIs for inhibiting bacterial enzymes. We also propose the combined use of ChoKIs and nanoparticles for targeted delivery to the pathogen while shielding the human host from any possible side effects of the inhibitors. More research should focus on the verification of putative bacterial ChoK activities and the characterization of ChoKIs with active enzymes. In conclusion, the presence of ChoK in a wide range of pathogenic bacteria and the distinct function of this enzyme has made it an attractive drug target. This review highlighted the possibility of "choking" bacterial ChoKs by using human ChoKIs.
  15. Raikundalia S, Sa'Dom SAFM, Few LL, Too WCS
    Oncol Lett, 2021 Mar;21(3):183.
    PMID: 33574922 DOI: 10.3892/ol.2021.12444
    Choline kinase (ChK) catalyzes the first step in the CDP-choline pathway for the synthesis of phosphatidylcholine. The α isoform of this enzyme is overexpressed in various types of cancer and its inhibition or downregulation has been applied as an anticancer strategy. In spite of increasing attention being paid to ChK expression, as well as its activity and inhibition in cancer, there are only limited studies available on the regulation of ChK, including its regulation by microRNAs (miRNAs/miRs). The dysregulation of gene expression by miRNAs is a common cause for carcinogenesis. In the present study, miR-367-3p was predicted to target the 3'-untranslated region (UTR) of the ChK α (chka) mRNA transcript. The binding of miR-367-3p to the 3'-UTR of chka was validated by a luciferase assay. The effects of the miR-367-3p mimic on chka gene and protein expression levels were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. miR-367-3p significantly downregulated the expression of chka to ~60% of the negative control. Cells transfected with miR-367-3p exhibited higher levels of apoptosis and a lower cell migration compared with the control. To the best of our knowledge, the present study provided the first experimental evidence of the regulation of chka expression by miR-367-3p. The pro-apoptotic and suppressive effects of miR-367-3p on cell migration were similar to the anticancer effects resulting from the inhibition of ChK enzyme activity or the knockdown of chka gene expression by small interfering RNA. Therefore, these findings may potentially lead to the use of miR-367-3p in anticancer strategies that target ChK.
  16. Mohamed Sa'dom SAF, Raikundalia S, Shamsuddin S, See Too WC, Few LL
    Genes (Basel), 2021 06 01;12(6).
    PMID: 34205960 DOI: 10.3390/genes12060853
    Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between -225 and -56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.
  17. Khalifa M, Few LL, Too WCS
    Mini Rev Med Chem, 2021 Dec 13.
    PMID: 34961459 DOI: 10.2174/1389557521666211213160256
    BACKGROUND: Pseudomonas aeruginosa is one of the most prevalent opportunistic pathogens in humans that has thrived and proved to be difficult to control in this "post-antibiotic era." Antibiotic alternatives are necessary for fighting against this resilient bacterium. Even though phages might not be "the wonder drug" that solves everything, they still provide a viable option to combat P. aeruginosa and curb the threat it imposes.

    MAIN FINDINGS: The combination of antibiotics with phages, however, poses a propitious treatment option for P. aeruginosa. Choline kinase (ChoK) is the enzyme that synthesizes phosphorylcholine subsequently incorporated into lipopolysaccharide located at the outer membrane of gram-negative bacteria. Recently, inhibition of ChoKs has been proposed as a promising antibacterial strategy. Successful docking of Hemicholinium-3, a choline kinase inhibitor, to the model structure of P. aeruginosa ChoK also supports the use of this inhibitor or its derivatives to inhibit the growth of this microorganism.

    CONCLUSION: Therefore, the combination of the novel antimicrobial "choline kinase inhibitors (ChoKIs)" with a phage cocktail or synthetic phages as a potential treatment for P. aeruginosa infection has been proposed.

  18. Chang CH, Few LL, Lim BH, Yvonne-Tee GB, Chew AL, See Too WC
    Parasitol Res, 2023 Jul;122(7):1651-1661.
    PMID: 37202563 DOI: 10.1007/s00436-023-07869-5
    The de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica is largely dependent on the CDP-choline and CDP-ethanolamine pathways. Although the first enzymes of these pathways, EhCK1 and EhCK2, have been previously characterized, their enzymatic activity was found to be low and undetectable, respectively. This study aimed to identify the unusual characteristics of these enzymes in this deadly parasite. The discovery that EhCKs prefer Mn2+ over the typical Mg2+ as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn2+, the activity of EhCK1 increased by approximately 108-fold compared to that in Mg2+. Specifically, in Mg2+, EhCK1 exhibited a Vmax and K0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn2+, it displayed a Vmax of 149.1 ± 2.5 U/mg and a K0.5 of 9.5 ± 0.1 mM. Moreover, when Mg2+ was present at a constant concentration of 12 mM, the K0.5 value for Mn2+ was ~ 2.4-fold lower than that in Mn2+ alone, without affecting its Vmax. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn2+, it is worth noting that its Km for choline and ATP were higher than in equimolar of Mg2+ in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn2+, exhibiting Michaelis-Menten kinetic with ethanolamine (Km = 312 ± 27 µM) and cooperativity with ATP (K0.5 = 2.1 ± 0.2 mM). Additionally, we investigated the effect of metal ions on the substrate recognition of human choline and ethanolamine kinase isoforms. Human choline kinase α2 was found to absolutely require Mg2+, while choline kinase β differentially recognized choline and ethanolamine in Mg2+ and Mn2+, respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn2+ binding, while Lys233 was essential for substrate catalysis but not metal ion binding. Overall, these findings provide insight into the unique characteristics of the EhCKs and highlight the potential for new approaches to treating amoebiasis. Amoebiasis is a challenging disease for clinicians to diagnose and treat, as many patients are asymptomatic. However, by studying the enzymes involved in the CDP-choline and CDP-ethanolamine pathways, which are crucial for de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica, there is great potential to discover new therapeutic approaches to combat this disease.
  19. Teh ZH, Lim BH, See Too WC, Few LL
    Trop Biomed, 2023 Dec 01;40(4):430-438.
    PMID: 38308830 DOI: 10.47665/tb.40.4.008
    Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.
  20. Chang CH, See Too WC, Lim BH, Few LL
    Acta Parasitol, 2024 Jan 03.
    PMID: 38172465 DOI: 10.1007/s11686-023-00763-1
    PURPOSE: Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future.

    METHODOLOGY: E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes.

    RESULTS: EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1 µmol/min/mg. Its Km for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting.

    CONCLUSIONS: With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

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