BACKGROUNDPyruvate phosphate dikinase (PPDK) reversibly catalyzes the interconversion of phosphoenolpyruvate (PEP) and pyruvic acid, leading to catabolism and adenosine triphosphate (ATP) synthesis or gluconeogenesis and ATP consumption. Molecular modeling of PPDKs from divergent organisms demonstrates that the orientation of the phosphorylatable histidine residue within the central domain of PPDK determines whether this enzyme promotes catabolism or gluconeogenesis. The goal of this study was to determine whether PDDK from Giardia underwent adaptive evolution in order to produce more energy under anaerobic conditions.

METHODSA total of 123 PPDK sequences from protozoans, proteobacteria, plants, and algae were selected, based upon sequence similarities to Giardia lamblia PPDK and Zea mays PPDK. Three-dimensional (3-D) models were generated for PPDKs from divergent organisms and were used to compare the orientation of the phosphorylatable histidine residue within the central domain of PPDKs. These PPDKs were compared using a maximum-likelihood tree.

RESULTSFor PPDK from Giardia, as well as from other anaerobic protozoans, the central domain tilted toward the N-terminal nucleotide-binding domain, indicating that this enzyme catalyzed ATP synthesis. Furthermore, the orientation of this central domain was determined by interactions between the N- and C-terminal domains. Phylogenetic analysis of the N- and C-terminal sequences of PPDKs from different species suggested that PPDK has likely undergone adaptive evolution in response to differences in environmental and metabolic conditions.

CONCLUSIONThese results suggested that PPDK in anaerobic organisms is functionally adapted to generate energy more efficiently in an anaerobic environment.

Adenosine Triphosphate ; metabolism ; Evolution, Molecular ; Giardia lamblia ; enzymology ; Protozoan Proteins ; chemistry ; classification ; genetics ; Pyruvate, Orthophosphate Dikinase ; chemistry ; classification ; genetics

OBJECTIVETo confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries.

METHODSGenomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank.

RESULTSOf nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3)) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world.

CONCLUSIONGenotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

Amino Acid Sequence ; Animals ; Base Sequence ; DNA, Protozoan ; chemistry ; Genotype ; Giardia lamblia ; classification ; enzymology ; genetics ; Polymerase Chain Reaction ; Restriction Mapping ; Triose-Phosphate Isomerase ; chemistry ; genetics

The cyclophilins (Cyps) are family members of proteins that exhibit peptidylprolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosprin A (CsA) in varying degrees. During the process of random sequencing of a cDNA library made from Giardia intestinalis WB strain, the cyclophilin gene (gicyp 1) was isolated. An open reading frame of gicyp 1 gene was 576 nucleotides, which corresponded to a translation product of 176 amino acids (Gicyp 1). The identity with other Cyps was about 58-71%. The 13 residues that constituted the CsA binding site of human cyclophilin were also detected in the amino acid sequence of Gicyp 1, including tryptophan residue essential for the drug binding. The single copy of the gicyp 1 gene was detected in the G. intestinalis chromosome by southern hybridization analysis. Recombinant Gicyp 1 protein clearly accelerated the rate of cis-- Amino Acid Sequence ; Animals ; Cloning, Molecular ; Cyclophilins/antagonists & inhibitors/chemistry/genetics/*isolation&purification ; Cyclosporine/metabolism/pharmacology ; Giardia lamblia/*chemistry/genetics ; Human ; Immunosuppressive Agents ; Molecular Sequence Data ; Protein Binding ; Protozoan Proteins ; Recombinant Proteins

OBJECTIVETo investigate the intraspecific difference of the triose phosphate isomerase (tim) gene from Giardia lamblia (G. lamblia).

METHODSTotal genomic DNA of G. lamblia was extracted and partial fragments of the triose phosphate isomerase (tim) gene were amplified by polymerase chain reaction (PCR). All nucleotide sequences were analyzed by using a phylogenetic analysis, which was constructed with parsimony and Neighbor-joining (N-J) methods.

RESULTSA total of 124 variable sites (23% of all sequences detected) was defined, most of which were found at the silent sites of codons. Two similar phylogenetic trees were constructed, subdividing 16 Giardia isolates into two groups.

CONCLUSIONThe genetic diversity of G. lamblia appeared to be little affected by factors of both host and geography, while natural-selection played an important role in DNA molecular evolution level of the tim gene. The tim gene may be considered a very useful genetic marker of the population genetic structure of G. lamblia.

Animals ; Base Sequence ; DNA, Protozoan ; chemistry ; genetics ; Giardia lamblia ; enzymology ; genetics ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Species Specificity ; Triose-Phosphate Isomerase ; genetics

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and beta,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.
Animals ; Cryptosporidium/*isolation & purification ; Cytoskeletal Proteins/genetics ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Genes, rRNA ; Giardia lamblia/*isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Portugal ; Protozoan Proteins/genetics ; RNA, Protozoan/genetics ; RNA, Ribosomal, 18S/genetics ; Risk Assessment ; Sequence Analysis, DNA ; Water/*parasitology

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.
Animals ; Cat Diseases/*parasitology ; Cats ; China ; Cluster Analysis ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Feces/parasitology ; Giardia lamblia/*classification/cytology/genetics/*isolation & purification ; Giardiasis/parasitology/*veterinary ; Microscopy ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/genetics ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.
Animals ; Cat Diseases/*parasitology ; Cats ; China ; Cluster Analysis ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Feces/parasitology ; Giardia lamblia/*classification/cytology/genetics/*isolation & purification ; Giardiasis/parasitology/*veterinary ; Microscopy ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/genetics ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.
Animals ; China ; Cluster Analysis ; Coinfection/parasitology/veterinary ; Cytoskeletal Proteins/genetics ; DNA, Protozoan/chemistry/genetics ; Dog Diseases/parasitology ; Dogs ; Genotype ; Giardia lamblia/*classification/*genetics/isolation & purification ; Giardiasis/parasitology/*veterinary ; Glutamate Dehydrogenase/genetics ; Molecular Sequence Data ; *Multilocus Sequence Typing ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 18S/genetics ; Triose-Phosphate Isomerase/genetics