Egbert Tannich

Metronidazole resistance in the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase.

To obtain insight into the mechanism of metronidazole resistance in the protozoan parasite Entamoeba histolytica, amoeba trophozoites were selected in vitro by stepwise exposures to increasing amounts of metronidazole, starting with sublethal doses of 4 μm. Subsequently, amoebae made resistant were able to continuously multiply in the presence of a 40 μm concentration of the drug. In contrast to mechanisms of metronidazole resistance in other protozoan parasites, resistant amoebae did not substantially down-regulate pyruvate:ferredoxin oxidoreductase or up-regulate P-glycoproteins, but exhibited increased expression of iron-containing superoxide dismutase (Fe-SOD) and peroxiredoxin and decreased expression of flavin reductase and ferredoxin 1. Episomal transfection and overexpression of the various antioxidant enzymes revealed significant reduction in susceptibility to metronidazole only in those cells overexpressing Fe-SOD. Reduction was highest in transfected cells simultaneously overexpressing Fe-SOD and peroxiredoxin. Although induced overexpression of Fe-SOD did not confer metronidazole resistance to the extent found in drug-selected cells, transfected cells quickly adapted to constant exposures of otherwise lethal metronidazole concentrations. Moreover, metronidazole selection of transfected amoebae favored retention of the Fe-SOD-containing plasmid. These results strongly suggest that peroxiredoxin and, in particular, Fe-SOD together with ferredoxin 1 are important components involved in the mechanism of metronidazole resistance in E. histolytica.

ENTAMOEBA HISTOLYTICA AND ENTAMOEBA DISPAR: DIFFERENCES IN NUMBERS AND EXPRESSION OF CYSTEINE PROTEINASE GENES

In order to identify molecules that might be responsible for the difference in pathogenicity between the two closely related protozoan parasites Entamoeba histolytica and Entamoeba dispar, we focussed on cysteine proteinases because this class of enzymes has been considered important for pathogenic tissue destruction. By screening a genomic library derived from an E. histolytica isolate, a total of six distinct genes (ehcp1–ehcp6) encoding typical prepro‐forms of cysteine proteinases were identified which differed from each other by 40% to 85% of their nucleotide sequences. Three of these genes, ehcp1, ehcp2, and ehcp5, which exhibited high levels of expression, were found to be responsible for approximately 90% of cysteine proteinase transcripts, whereas the remaining three were either not or only marginally expressed. Expression of the different genes directly correlated with the level of activity of the respective enzymes in trophozoite lysates. Purification of the enzymes and N‐terminal sequencing revealed that virtually all cysteine proteinase activity of E. histolytica can be attributed to three enzymes namely EhCP1, EhCP2 and EhCP5. Southern blot analysis indicated that just two of these abundantly expressed genes are missing in E. dispar. On the other hand, genes analogous to four of the six genes identified in E.histolytica were found to be present in E. dispar, but only two of these are expressed within the trophozoite stage.

The Intestinal Protozoan Parasite Entamoeba histolytica Contains 20 Cysteine Protease Genes, of Which Only a Small Subset Is Expressed during In Vitro Cultivation

ABSTRACT Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.

Structure and Content of the Entamoeba histolytica Genome

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore bacterial cytoplasmic membranes

Three peptides with pore‐forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed‐phase high‐performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic α‐helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore‐forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram‐positive bacteria. The amoebapores represent a distinct family of membrane‐active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.

Real-Time PCR for Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar in Fecal Samples

ABSTRACT A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica- or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.

Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica

Major pathogenic functions of Entamoeba histolytica involved in destruction of host tissues are the degradation of extracellular matrix proteins mediated by secreted cysteine proteinases and contact‐dependent killing of host cells via membrane‐active factors. A soluble protein with an affinity for membranes was purified from amoebic extracts to apparent homogeneity. N‐terminal sequencing and subsequent molecular cloning of the factor revealed that it is a member of the cysteine proteinase family of E. histolytica, which we termed CP5. Further experiments with the purified protein showed that it has potent proteolytic activity that is abrogated in the presence of inhibitors specific for cysteine proteinases. The enzyme firmly associates with membranes retaining its proteolytic activity and it produces cytopathic effects on cultured monolayers. A model of the three‐dimensional structure of CP5 revealed the presence of a hydrophobic patch that may account for the potential of the protein to associate with membranes. Immunocytochemical localization of the enzyme to the surface of the amoeba in combination with the recent finding that the gene encoding CP5 is missing in the closely related but non‐pathogenic Entamoeba dispar suggests a potential role of the protein in host tissue destruction of E. histolytica.

Longitudinal Study of Intestinal Entamoeba histolytica Infections in Asymptomatic Adult Carriers

ABSTRACT To gain insight into the dynamics of intestinal Entamoeba histolytica infection, a longitudinal study was performed over an observation period of 15 months with a group of 383 randomly selected adult individuals (mean age, 38.5 years) living in an area of amebiasis endemicity in central Vietnam. Ameba infection was diagnosed by using species-specific PCR and DNA extracted directly from fecal samples. The results indicated an E. histolytica prevalence of 11.2% and an annual new infection rate of 4.1% in the study population. Follow-up of the 43 individuals who were E. histolytica positive at enrollment suggested a regular exponential decline in infection of about 3% per month and a mean half-life of infection of more than 15 months. However, the reinfection rate for this group of participants was 2.7 times higher than that predicted for the study population as a whole. Both the reappearance of the parasite after successful treatment of E. histolytica infection and changes in “genetic fingerprints” of parasites during the course of infection revealed an annual new infection rate of about 11.5%. Thus, the mean half-life of E. histolytica infection was calculated to be 12.9 months (95% confidence interval, 10.2 to 15.6 months). Notably, none of the participants developed symptoms compatible with invasive intestinal amebiasis, and only one of the subjects developed an amebic liver abscess during the observation period.

Two cases of autochthonous Plasmodium falciparum malaria in Germany with evidence for local transmission by indigenous Anopheles plumbeus

Autochthonous Plasmodium falciparum malaria (PFM) in Central Europe has been reported repeatedly, transmission of the parasite being attributed to blood transfusion or imported P. falciparum‐infected vectors. We report two cases of PFM in German children without travel history to malaria‐endemic areas. Both infections occurred during a stay in a hospital where a child from Angola with chronic P. falciparum infection was hospitalized at the time. Known routes of transmission, such as imported mosquitoes or blood transfusion, were very unlikely or could be excluded, whereas evidence was obtained for transmission by the indigenous mosquito species Anopheles plumbeus.

Pathogenic and nonpathogenicEntamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase

Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytica isolate HM-1:IMSS. Under anaerobic culture conditions, 18.7 (+/- 4.9) units SOD activity (mg protein)-1 were found. By inhibition studies the activity was attributed to an iron-containing type of SOD (FeSOD). Using degenerate oligonucleotide primers derived from regions highly conserved in prokaryotic FeSOD sequences, a genomic DNA fragment was amplified by the polymerase chain reaction. The fragment was used to isolate FeSOD specific cDNA clones from a pathogenic and a nonpathogenic E. histolytica isolate. A comparison of the 2 sequences revealed 5% nucleotide differences resulting in a single amino acid exchange. The primary structure showed the characteristics of an iron-containing type of SOD with a homology of approximately 55% with other FeSOD sequences. The enzyme was found to be encoded by single copy genes in both the pathogenic and the nonpathogenic E. histolytica, but restriction fragment lengths differed between the 2 groups. In 5 isolates studied, no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA.