John P. Ackers

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. histolyticas 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.

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 In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

IMMUNOLOGICAL DIFFERENTIATION OF PATHOGENIC AND NON-PATHOGENIC ISOLATES OF ENTAMOEBA HISTOLYTICA

Entamoeba histolytica can act as a harmless commensal organism in the lumen of the large intestine, or can cause invasive amoebiasis. Some workers have suggested that there are two distinct subspecies of this organism, and that only one of these is associated with invasive disease. Present isoenzyme tests to identify the subspecies take several days to analyse: we report a technique that uses immunofluorescence with monoclonal antibodies, takes two days to perform, and may, therefore, assist in the clinical management of patients infected with this organism.

A novel transcribed repeat element from Entamoeba histolytica

We have identified an unusual 0.55-kb DNA repeat element specific to Entamoeba histolytica (Eh) which we call interspersed element (IE). The IE is a common feature in independently isolated genomic and cDNA fragments. Hybridization of labeled IE sequences to trophozoite DNA, RNA and first-strand cDNA prepared from poly(A)-enriched mRNA indicate that the IE are reiterated about 500 times per Eh trophozoite and that one or more can be found as RNA transcripts. These features and the degree of conservation of IE suggest a possible role for these sequences.

Absence of lipophosphoglycan-like glycoconjugates in Entamoeba dispar

Invasive amoebiasis is the result of infection of Entamoeba histolytica. The closely related Entamoeba dispar can colonize the human gut but does not cause invasive disease. In this study, E. dispar was analysed for the presence of the lipophosphoglycan-like (LPG) glycoconjugate known to be present on the cell surface of E. histolytica. E. dispar cells were radio-isotope labelled with [3H]galactose or [3H]inositol. The acidic glycoconjugates were extracted and analysed by hydrophobic chromatography over phenyl-Sepharose and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No LPG-like molecules could be identified in E. dispar in contrast to E. histolytica, suggesting that these molecules may be absent in the non-pathogenic species.

Interaction between Trichomonas vaginalis and other pathogenic micro-organisms of the human genital tract.

Trichomonas vaginalis organisms were mixed with suspensions of Neisseria gonorrhoeae, Mycoplasma hominis or Chlamydia trachomatis to allow ingestion of these micro-organisms by the trichomonads. Culture studies indicated that gonococci and mycoplasmas were ingested and that the number of intracellular viable organisms decreased rapidly, most gonococci being killed within six hours and all mycoplasmas within three hours. Electron microscopy revealed phagocytic uptake and destruction of these two micro-organisms within the trichomonads, gonococcal degradation being associated with lysosomal enzyme activity. There was no evidence from cultural or electron microscopy studies that C trachomatis organisms persisted in mixed culture with T vaginalis.

Evaluation of an enzyme-linked immunosorbent assay for the detection of antibody to Trichomonas vaginalis in sera and vaginal secretions.

Using a whole-cell antigen antibody to Trichomonas vaginalis was measured by an enzyme-linked immunosorbent assay (ELISA). IgG antibody was found in sera from only three of 99 children under 12 years of age. In contrast, serum IgG or IgM antibody or both were detected in 80.4% of women who had vaginal trichomoniasis and in 13.7% of uninfected women. Although antibody was found in cervical and vaginal secretions, the correlation between current infection and the presence of antibody was poorer than found between circulating antibody and infection. IgG or IgA antibody or both was detected in the secretions of 73.2% and 41% of infected and uninfected women respectively. This may be accounted for, at least partly, by previous infections since antibody, circulating or local, was found most often in women who had a history of trichomoniasis. There was no indication that some other vaginal micro-organism stimulated antibody directed against T vaginalis.

Trichomonas vaginalis infection

The organism Trichomonas vaginalis is a sexually transmissible protozoal parasite. It is the commonest curable sexually transmitted infection (STI); The World Health Organization estimates that about 170 million new cases occur annually.1 It is a common cause of vaginal discharge in women, in whom it may also cause vulval irritation and inflammation, dysuria, and inflammation of the exo-cervix. It has been associated with dysuria and urethral discharge in men but asymptomatic infection also occurs in both sexes. T vaginalis infection is associated with low socioeconomic status, and is more prevalent in developing than in developed countries.2,3 Opinions vary concerning whether or not T vaginalis can be transmitted by non-sexual contact.4,5 A morphologically similar organism, Pentatrichomonas hominis , is a commensal of the human large intestine, but conventional wisdom has it that this organism does not multiply in the human reproductive tract. Microscopy of a wet mount preparation is the most commonly used diagnostic test for T vaginalis infection. Characteristic motile flagellated protozoa are readily seen. Microscopy for T vaginalis should be performed as soon as possible after the sample is taken as motility diminishes with time. Wet …

The Antigen of Bordetella pertussis that Induces Bactericidal Antibody and its Relationship to Protection of Mice

SUMMARY: The ability to protect mice against intracerebral infection and to elicit, after one dose, complement-mediated bactericidal antibody capable of killing a mouse-virulent phase I strain in vitro, was tested in seven cultures of Bordetella pertussis: three typical phase I strains; two strains grown in the presence of 0.5 mg nicotinic acid/ml; one phase IV strain; and the atypical strain 134. The typical strains showed both activities: the nicotinic acid-grown and phase IV strains elicited high antibody titres, but did not confer protection, whereas strain 134 protected, but did not elicit bactericidal antibody. Pyrogenic material extracted from dried organisms by hot phenol and water (lipopolysaccharide) was not antigenic in mice; however, with all strains, except 134, multiple doses of this material coupled to stromata or Escherichia coli conjugated protein elicited bactericidal and precipitating antisera in mice, although the mice were not protected. Strain 134 contained approximately one quarter of the normal amount of endotoxin, as assayed in actinomycin D-sensitized mice, but was as effective as normal strains as an adjuvant for haemolysin production.

Ribosomal DNA sequences in the differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica.

Recombinant ribosomal DNA sequences were amplified by PCR and used as probes to perform a fingerprint analysis of total DNA from different Entamoeba histolytica isolates. RFLPs obtained with one of the probes, R-1, support previous proposals that pathogenic and non-pathogenic E. histolytica are closely related, yet genotypically distinct. Another probe, R-2, while not distinguishing between the two forms of E. hystolytica, was able to differentiate between them and E. moshkovskii, which has morphologically identical cysts and trophozoites. A third probe, BR-1, identified strain-specific RFLPs.