Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes

Entamoeba histolytica Phosphoserine aminotransferase (EhPSAT): insights into the structure-function relationship

<p>Abstract</p> <p>Background</p> <p>Presence of phosphorylated Serine biosynthesis pathway upstream to the de novo cysteine biosynthesis pathway makes PSAT a crucial enzyme. Besides this, phoshoserine produced by the enzyme can also be taken up directly by cysteine synthase as a substrate. PSAT is a PLP dependent enzyme where the cofactor serves as an epicenter for functional catalysis with the active site architecture playing crucial role in optimum function of the enzyme.</p> <p>Findings</p> <p>EhPSAT is a homodimer of molecular mass 86 kDa. To understand the structural modulations associated with pH dependent changes in functional activity of EhPSAT detailed biophysical studies were carried out. pH alterations had no significant effect on the secondary structure, cofactor orientation and oligomeric configuration of the enzyme however, pH dependent compaction in molecular dimensions was observed. Most interestingly, a direct correlation between pH induced modulation of functional activity and orientation of Trp 101 present in the active site of the enzyme was observed. Sodium halides nullified the pH induced global changes in the enzyme, however differential effect of these salts on the active site microenvironment and functional activity of the enzyme was observed.</p> <p>Conclusions</p> <p>The study unequivocally demonstrates that pH induced selective modification of active site microenvironment and not global change in structure or oligomeric status of the enzyme is responsible for the pH dependent change in enzymatic activity of PSAT.</p

Metabolic Profiling of the Protozoan Parasite Entamoeba invadens Revealed Activation of Unpredicted Pathway during Encystation

Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst). We also found remarkable induction of γ-aminobutyric acid (GABA) during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause

Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches

Our current taxonomic perspective on Entamoeba is largely based on small-subunit ribosomal RNA genes (SSU rDNA) from Entamoeba species identified in vertebrate hosts with minor exceptions such as E. moshkovskii from sewage water and E. marina from marine sediment. Other Entamoeba species have also been morphologically identified and described from non-vertebrate species such as insects; however, their genetic diversity remains unknown. In order to further disclose the diversity of the genus, we investigated Entamoeba spp. in the intestines of three cockroach species: Periplaneta americana, Blaptica dubia, and Gromphadorhina oblongonota. We obtained 134 Entamoeba SSU rDNA sequences from 186 cockroaches by direct nested PCR using the DNA extracts of intestines from cockroaches, followed by scrutinized BLASTn screening and phylogenetic analyses. All the sequences identified in this study were distinct from those reported from known Entamoeba species, and considered as novel Entamoeba ribosomal lineages. Furthermore, they were positioned at the base of the clade of known Entamoeba species and displayed remarkable degree of genetic diversity comprising nine major groups in the three cockroach species. This is the first report of the diversity of SSU rDNA sequences from Entamoeba in non-vertebrate host species, and should help to understand the genetic diversity of the genus Entamoeba

Cysteine protease-binding protein family 6 mediates the trafficking of amylases to phagosomes in the enteric protozoan entamoeba histolytica

金沢大学医薬保健研究域薬学系Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or α-hexosaminidase β-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to β-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins. © 2013, American Society for Microbiology

Novel transmembrane receptor involved in phagosome transport of lysozymes and β-hexosaminidase in the enteric protozoan Entamoeba histolytica

金沢大学医薬保健研究域薬学系Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes. © 2012 Furukawa et al

Molecular evidence for zoonotic transmission of Giardia duodenalis among dairy farm workers in West Bengal, India

No study in the past has examined the genetic diversity and zoonotic potential of Giardia duodenalis in dairy cattle in India. To assess the importance of these animals as a source of human G. duodenalis infections and determine the epidemiology of bovine giardiasis in India, fecal samples from 180 calves, heifers and adults and 51 dairy farm workers on two dairy farms in West Bengal, India were genotyped by PCR-RFLP analysis of the β-giardin gene of G. duodenalis followed by DNA sequencing of the nested PCR products. The overall prevalence of G. duodenalis in cattle was 12.2% (22/180), the infection being more prevalent in younger calves than in adult cattle. Zoonotic G. duodenalis Assemblage A1 was identified in both calves and workers although the most prevalent genotype detected in cattle was a novel Assemblage E subgenotype. These findings clearly suggest that there is a potential risk of zoonotic transmission of G. duodenalis infections between cattle and humans on dairy farms in India

Trend of Entamoeba histolytica infestation in Kolkata

Background: Entamoeba histolytica infection is found almost all over the world and is highly endemic and a major cause of parasitic diarrhoea particularly in the developing countries. Methods: A systemic surveillance was set up at the Infectious Disease hospital, Kolkata, India between November 2007 and October 2009 for understanding the trend of E. histolytica infection in Kolkata. Fecal samples were collected from diarrhoeal patients attending the hospital, under the surveillance system and processed for detection of E. histolytica. Results: During the last two years about 2500 diarrhoeal samples were collected and screened for E. histolytica. About 3.6% were positive for E. histolytica. As compared to the earlier years, E. histolytica infection was observed to be less amongst patients screened during the last two years. No seasonality was observed in Kolkata although in the neighboring tropical country Bangladesh, a typical seasonality of E. histolytica infection was noticed. Conclusion: The study indicates that the detection rate of E. histolytica infection amongst diarrhoeal patients in Kolkata is decreasing during the last two years than that of Bangladesh

A Key Role for Old Yellow Enzyme in the Metabolism of Drugs by Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas' disease. So far, first choice anti-chagasic drugs in use have been shown to have undesirable side effects in addition to the emergence of parasite resistance and the lack of prospect for vaccine against T. cruzi infection. Thus, the isolation and characterization of molecules essential in parasite metabolism of the anti-chagasic drugs are fundamental for the development of new strategies for rational drug design and/or the improvement of the current chemotherapy. While searching for a prostaglandin (PG) F2α synthase homologue, we have identified a novel “old yellow enzyme” from T. cruzi (TcOYE), cloned its cDNA, and overexpressed the recombinant enzyme. Here, we show that TcOYE reduced 9,11-endoperoxide PGH2 to PGF2α as well as a variety of trypanocidal drugs. By electron spin resonance experiments, we found that TcOYE specifically catalyzed one-electron reduction of menadione and β-lapachone to semiquinone-free radicals with concomitant generation of superoxide radical anions, while catalyzing solely the two-electron reduction of nifurtimox and 4-nitroquinoline-N-oxide drugs without free radical production. Interestingly, immunoprecipitation experiments revealed that anti-TcOYE polyclonal antibody abolished major reductase activities of the lysates toward these drugs, identifying TcOYE as a key drug-metabolizing enzyme by which quinone drugs have their mechanism of action