Replacement of the essential Dictyostelium Arp2 gene by its Entamoeba homologue using parasexual genetics

<p>Abstract</p> <p>Background</p> <p>Cell motility is an essential feature of the pathogenesis and morbidity of amoebiasis caused by <it>Entamoeba histolytica</it>. As motility depends on cytoskeletal organisation and regulation, a study of the molecular components involved is key to a better understanding of amoebic pathogenesis. However, little is known about the physiological roles, interactions and regulation of the proteins of the <it>Entamoeba </it>cytoskeleton.</p> <p>Results</p> <p>We have established a genetic strategy that uses parasexual genetics to allow essential <it>Dictyostelium discoideum </it>genes to be manipulated and replaced with modified or tagged homologues. Our results show that actin related protein 2 (Arp2) is essential for survival, but that the <it>Dictyostelium </it>protein can be complemented by <it>E. histolytica </it>Arp2, despite the presence of an insertion of 16 amino acids in an otherwise highly conserved protein. Replacement of endogenous Arp2 with <it>myc</it>-tagged <it>Entamoeba </it>or <it>Dictyostelium </it>Arp2 has no obvious effects on growth and the protein incorporates effectively into the Arp2/3 complex.</p> <p>Conclusion</p> <p>We have established an effective two-step method for replacing genes that are required for survival. Our protocol will allow such genes to be studied far more easily, and also allows an unambiguous demonstration that particular genes are truly essential. In addition, cells in which the <it>Dictyostelium </it>Arp2 has been replaced by the <it>Entamoeba </it>protein are potential targets for drug screens.</p

Genotyping of Entamoeba species in South Africa: diversity, stability, and transmission patterns within families.

Using a recently described polymerase chain reaction-based DNA typing method for Entamoeba histolytica and E. dispar, we investigated the genetic diversity of these species in a geographically restricted region of South Africa. Patterns were stable over time in the same infection, and, with few exceptions, infected family members carried the same strain. However, both species exhibited remarkable variation, with no 2 family groups being infected with the same strain of E. histolytica. Mixed infections were rare. The results indicate that this typing method will be useful in identifying epidemiological linkage between infections

Characterisation of polymorphic DNA and its application to typing of Entamoeba histolytica and Entamoeba dispar

A key question in amoebiasis is whether the variable symptoms of amoebic infections are a reflection of different strains of E. histolytica. To address this, tools that allow typing of E. histolytica isolates are needed and this objective formed the basis of the present study. A method for PCR-based DNA typing of E. histolytica isolates has been developed using multiple loci with internal short tandem repeats (STRs) as the polymorphic markers. It has been shown that E. dispar isolates can also be typed by this approach and species-specific primers have been developed for two loci. E. histolytica and E. dispar samples from a wide geographic range were studied to validate the general utility of these loci. Results revealed that E. histolytica is genetically highly variable. This was evident in all the communities studied. E. dispar also displays intra-species variation. The patterns seen for individual strains of both species were stable over time in the same infection. With few exceptions a single E. histolytica or E. dispar strain was identified in samples from infected family groups and outbreaks. Our results show both the existence of mixed species infection as well as the possibility of co-infection with different strains of the same species

Simultaneous differentiation and typing of Entamoeba histolytica and Entamoeba dispar.

Sequences corresponding to some of the polymorphic loci previously reported from Entamoeba histolytica have been detected in Entamoeba dispar. Comparison of nucleotide sequences of two loci between E. dispar strain SAW760 and E. histolytica strain HM-1:IMSS revealed significant differences in both repeat and flanking regions. The tandem repeat units varied not only in sequence but also in number and arrangement between the two species at both the loci. Using the sequences obtained, primer pairs aimed at amplifying species-specific products were designed and tested on a variety of E. histolytica and E. dispar samples. Amplification results were in complete agreement with the original species classification in all cases, and the PCR products displayed discernible size and pattern variations among the isolates

Unique organisation of tRNA genes in Entamoeba histolytica.

The genome sequence of the protistan parasite Entamoeba histolytica HM-1:IMSS has been completed recently. Among the findings has been a unique organisation for the tRNA genes in this organism. Forty-two of the tRNA isoacceptor types are encoded in tandem arrays that vary in unit length from 490 to 1775 basepairs and contain from 1 to 5 tRNA genes. In three cases a 5S RNA gene is also present in the unit. An estimated 10% of the genome is made up of these arrays. Interspersed between RNA-encoding sequences are short tandem repeats that are polymorphic between isolates and, in some cases, within isolates. The number and organisation of tRNA genes in E. histolytica is unprecedented. In addition to encoding the tRNAs of the organism we propose that the arrays may fulfil a structural role in the genome

Use of PCR Amplification of tRNA Gene-Linked Short Tandem Repeats for Genotyping Entamoeba histolytica

We have developed a reliable method for PCR-based genotyping of Entamoeba histolytica based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species. Species-specific primer pairs were designed that differentiate E. histolytica from E. dispar as well as that reveal intraspecies PCR product length polymorphisms. The primers were tested with samples from different parts of the world, and DNA was extracted from cultured cells as well as liver abscess pus and feces by various methods. We now have the tools necessary to investigate a possible link between parasite genotype and the outcome of infection with Entamoeba histolytica, as well as other aspects of the organism's epidemiology

Genetic diversity inEntamoeba histolytica

Genetic diversity within Entamoeba histolytica led to the re- description of the species 10 years ago. However, more recent investigation has revealed significant diversity within the re- defined species. Both protein-coding and non-coding sequences show variability, but the common feature in all cases is the presence of short tandem repeats of varying length and sequence. The ability to identify strains of E. histolytica may lead to insights into the population structure and epidemiology of the organism

The presence of antiamoebic constituents in psyllium husk

The crude extract of psyllium husk (ispaghula) and its active constituent (petroleum fraction) caused varying degrees of growth inhibition in three different species of Entamoeba, i.e. Entamoeba histolytica, E. invadens and E. dispar. The inhibitory effect of the crude extract was in the dose range of 1-10 mg/mL, whereas a similar inhibitory effect was obtained with the petroleum fraction at a much lower dose (0.1-1.0 mg/mL), indicating that the active chemical(s) is/are concentrated in the petroleum fraction. These data support the traditional use of psyllium husk in amoebic dysentery