Biomedical subjects

Ute Willhoeft

Publications and source records attributed to Ute Willhoeft.

5 recordsLinked to original sources

PubMed2005

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Animals↗

PubMed2005

Analysis of 5' junctions of human LINE-1 and Alu retrotransposons suggests an alternative model for 5'-end attachment requiring microhomology-mediated end-joining.

Insertion of the human non-LTR retrotransposon LINE-1 (L1) into chromosomal DNA is thought to be initiated by a mechanism called target-primed reverse transcription (TPRT). This mechanism readily accounts for the attachment of the 3'-end of an L1 copy to the genomic target, but the subsequent integration steps leading to the attachment of the 5'-end to the chromosomal DNA are still cause for speculation. By applying bioinformatics to analyze the 5' junctions of recent L1 insertions in the human genome, we provide evidence that L1 uses at least two distinct mechanisms to link the 5'-end of the nascent L1 copy to its genomic target. While 5'-truncated L1 elements show a statistically significant preference for short patches of overlapping nucleotides between their target site and the point of truncation, full-length insertions display no distinct bias for such microhomologies at their 5'-ends. In a second genome-wide approach, we analyzed Alu elements to examine whether these nonautonomous retrotransposons, which are thought to be mobilized through L1 proteins, show similar characteristics. We found that Alu elements appear to be predominantly integrated via a pathway not involving overlapping nucleotides. The results indicate that a cellular nonhomologous DNA end-joining pathway may resolve intermediates from incomplete L1 retrotransposition events and thus lead to 5' truncations.

Alu Elements↗

PubMed2004

Characterization of chitin synthases from Entamoeba.

A major component of the Entamoeba cyst wall is chitin, a homopolymer of beta-(1,4)-linked N-acetyl-D-glucosamine. Polymerization of chitin requires the presence of active chitin synthases (CHS), a group of enzymes belonging to the family of beta-glycosyl transferases. CHS have been described for fungi, insects, and nematodes; however, information is lacking about the structure and expression of this class of enzymes in protozoons such as Entamoeba. In this study, the primary structures of two putative E. histolytica CHS (EhCHS-1 and EhCHS-2) were determined by gene cloning and homologous proteins were identified in databases from E. dispar and the reptilian parasite E. invadens. The latter constitutes the widely used model organism for the study of Entamoeba cyst development. The two ameba enzymes revealed between 23% and 33% sequence similarity to CHS from other organisms with full conservation of all residues critically important for CHS activity. Interestingly, EhCHS-1 and EhCHS-2 differed substantially in their predicted molecular weights (73 kD vs. 114 kD) as well as in their isoelectric points (5.04 vs. 8.05), and homology was restricted to a central stretch of about 400 amino acid residues containing the catalytic domain. Outside the catalytic domain, EhCHS-1 was predicted to have seven transmembrane helices (TMH) of which the majority is located within the C-terminal part, resembling the situation found in yeast; whereas, EhCHS-2 is structurally related to nematode or insect chitin synthases, as it contained 17 predicted TMHs of which the majority is located within the N-terminal part of the molecule. Northern blot analysis revealed that genes corresponding to CHS-1 and CHS-2 are not expressed in Entamoeba trophozoites, but substantial amounts of CHS-1 and CHS-2 RNA were present 4 to 8 hours after induction of cyst formation by glucose deprivation of E. invadens. The time-courses of expression differed slightly between the two ameba CHS genes, as in contrast to CHS-1 RNA, expression of CHS-2 RNA was more transient and no plateau was observed between 8 and 16 hours of encystation. However, both CHS RNAs were no longer detectable after 48 hours when most of the cells had been transformed into mature cysts.

Amino Acid Sequence↗

PubMed2003

Pig complement regulator factor H: molecular cloning and functional characterization.

Complement is an efficient defense mechanism of innate immunity. Factor H is the central complement regulator of the alternative pathway, acting in the fluid-phase and on self surfaces. Pigs are considered a suitable source for xenotransplantation and thus several membrane-bound pig complement regulators with importance for the acute rejection phase have been investigated. However, pig fluid-phase regulators have not been described so far. We report the cloning, expression and functional characterization of pig factor H. After constructing a pig liver cDNA library, a full-length factor H cDNA was isolated and sequenced. The predicted protein is organized in 20 short consensus repeat (SCR) domains and has an overall identity of 62% to the human protein. For functional characterization, three deletion constructs of pig factor H were expressed in insect cells. Pig factor H construct SCR 1-4 has cofactor activity for factor I-mediated cleavage of human C3b, which is similar to the human regulator. In addition, this N-terminal construct binds to human C3b, while a construct consisting of SCR 15-20 showed a weaker binding to human C3b/C3d. Pig factor H has two major binding sites for heparin, as the two constructs representing SCR 1-7 and SCR 15-20 proteins, but not the SCR 1-4 protein, bind heparin. The C-terminal construct is able to bind to human endothelial cells, as assayed by FACS. We show that pig and human factor H share functional characteristics in complement regulation and cell surface binding. Possible consequences of using pig livers for xenotransplantation are discussed.

Amino Acid Sequence↗

PubMed2002

The abundant polyadenylated transcript 2 DNA sequence of the pathogenic protozoan parasite Entamoeba histolytica represents a nonautonomous non-long-terminal-repeat retrotransposon-like element which is absent in the closely related nonpathogenic species Entamoeba dispar.

While comparing gene expression in the pathogenic organism Entamoeba histolytica and the closely related but nonpathogenic species Entamoeba dispar, we discovered that the E. histolytica abundant polyadenylated transcript 2 (ehapt2) and corresponding genomic copies are absent in E. dispar. Although polyadenylated, ehapt2 does not contain any overt open reading frame. Southern blot and sequence analyses revealed that about 500 copies of ehapt2 genomic elements were present in each cell and that the copies were distributed throughout the ameba genome. The various ehapt2 elements are regularly located in the vicinity of protein-encoding genes, downstream of pyrimidine-rich sequence stretches (40 to 125 bp; CT content, 79.2 to 85.5%), and are flanked by duplicated target sites of variable length. Target site duplications were obviously generated during integration of ehapt2 into the E. histolytica genome as one copy of the flanking repeat and the complete ehapt2 element are specifically absent in orthologous E. dispar genomic sequences. ehapt2 shares 3' sequences with EhRLE, a recently identified non-long-terminal-repeat (non-LTR) retrotransposon-like element of E. histolytica, which contains a conceptual open reading frame for reverse transcriptase. Thus, ehapt2 has all of the properties of nonautonomous non-LTR retrotransposons. A comparison of various E. histolytica isolates suggested that transposition of ehapt2 takes place at a very low frequency as the genomic localization of ehapt2 elements was found to be well conserved. A mobile element such as ehapt2 could be a suitable mechanism to explain the infrequent and late transition of E. histolytica from a harmless gut commensal to an invasive pathogen.

Animals↗