DNA Probes for Acanthamoeba Genomics and Epidemiology
Principal Investigator: Paul Fuerst
Abstract: DESCRIPTION: This study extends previous investigations of DNA diagnostic approaches for the opportunistically pathogenic protist Acanthamoeba, the causative agent of the sight-threatening disease Acanthamoeba keratitis (AK). Prior work used nucleotide sequences of the small subunit ribosomal RNA (ssu rDNA) genes from the nucleus and mitochondria to determine the phylogenetic relationships of Acanthamoeba species/strains. These studies showed that the taxonomy of Acanthamoeba could be represented more reliably by "sequence types" (or genotypes) rather than by traditional approaches based on morphological characters. Importantly, results indicated that the vast majority of AK cases are associated with a specific Acanthamoeba genotype: T4. We have now identified genotypes associated with AK (AKpath), genotypes associated with other disease manifestations such as granulomatous amebic encephalitis (GAE) (NAKpath), and genotypes from environmental samples not found in infections (NONpath). Further, phylogenetic structure was observed within genotype T4, a genotype found in AK infections, non-AK infections, and the environment. To extend previous studies, we propose to identify protein-coding gene alleles that may be unique to the pathogenic genotypes of Acanthamoeba. First, we will identify sequence variation in three protein-coding genes (profilin [an actin binding protein], a serine protease-like enzyme, and actophorin [an actin depolymerizing factor]) that may be involved in the pathogenicity of the amoeba. Using known DNA sequences of these genes, PCR primers have been constructed to obtain DNA products from Acanthamoeba genotypes. The sequences of the products will be determined, and comparative analyses of variability will be performed. Allelic differences in genes potentially involved in pathogenicity will be correlated with the classification of pathogenic and non-pathogenic genotypes. The genes also represent an independent test of Acanthamoeba phylogenies that have been based on non-coding ssu rDNA genes. Preliminary results for one of these genes, profilin, supports its use in these studies. Second, we will determine the DNA sequence of a large portion (about 12kb) of the Acanthamoeba mitochondrial genome (designated mt-l) for different genotypes. Mitochondrial genes have been shown to play roles in pathogenicity in Acanthamoeba, and other organisms. First, we will examine variation in two genes (cytochrome oxidase I and II) located on this mitochondrial fragment. Comparative genomic analyses of the mt-l region will be performed for AKpath, NAKpath, and NONpath Acanthamoeba genotypes to test if sequence differences, or allelic variation of mitochondrial genes are correlated with pathogenicity. Next, other genes in the mt-l region will be analyzed. Since previous work has shown that the mitochondrial ssu rDNA distinguishes sub-lineages within the genotype associated with AK, the mt-l sequence from sub-lineages of T4 also will be determined for comparative analyses. Preliminary results show that this region can be amplified in disparate genotypes. Further, these results may provide potential targets for future treatment, since products of these genes may be antibiotic targets.
Funding Period: 1992-01-01 - 2009-08-31
more information: NIH RePORT
- Identification and distribution of Acanthamoeba species genotypes associated with nonkeratitis infectionsGregory C Booton
Department of Evolution, Ecology, Organismal Biology, The Ohio State University, 318 W 12th Ave, Columbus, OH 43210, USA
J Clin Microbiol 43:1689-93. 2005..However, the genotypes that were isolated from brains have not been observed in environmental isolates of Acanthamoeba, and their natural ecological niche is unknown...
- Genotypic identification of Acanthamoeba sp. isolates associated with an outbreak of acanthamoeba keratitisGregory C Booton
Departments of Evolution, Ecology, and Organismal Biology and daggerMolecular Genetics, The Ohio State University, Columbus, OH 43210, USA
Cornea 28:673-6. 2009..To determine whether increased rates of Acanthamoeba keratitis (AK) are due to changes in municipal water treatment or to emergence of a more pathogenic strain of Acanthamoeba...
- Survival of Acanthamoeba cysts after desiccation for more than 20 yearsRama Sriram
Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341 3724, USA
J Clin Microbiol 46:4045-8. 2008..It has two stages in its life cycle: a trophic stage and a resistant cyst stage. We describe here the ability of Acanthamoeba cysts to survive desiccation for more than 20 years...
- The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitisElmer Y Tu
Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612, USA
Cornea 27:764-72. 2008..To compare the relative diagnostic value of confocal microscopy and superficial corneal cultures in the diagnosis of Acanthamoeba keratitis by using clinical and microbiologic definitions of disease...
- Efficacy of contact lens systems against recent clinical and tap water Acanthamoeba isolatesMegan E Shoff
Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, Columbus, OH 43210, USA
Cornea 27:713-9. 2008....
- Prognostic factors affecting visual outcome in Acanthamoeba keratitisElmer Y Tu
University of Illinois at Chicago, Department of Ophthalmology, Chicago, Illinois, USA
Ophthalmology 115:1998-2003. 2008..To identify clinical and demographic factors associated with a worse visual outcome in Acanthamoeba keratitis (AK)...
- The association of contact lens solution use and Acanthamoeba keratitisCharlotte E Joslin
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA
Am J Ophthalmol 144:169-180. 2007..To investigate Acanthamoeba keratitis (AK) risk factors. Diagnosis of AK, a rare but serious corneal infection, has recently increased significantly at the University of Illinois at Chicago (UIC) Cornea Service...
- Infections caused by pathogenic free-living amebas (Balamuthia mandrillaris and Acanthamoeba sp.) in horsesHailu Kinde
California Animal Health and Food Safety Laboratory System CAHFS, San Bernardino Branch, 105 West Central Avenue, San Bernardino, CA 92408, USA
J Vet Diagn Invest 19:317-22. 2007..and/or Escherichia coli and Pseudomonas sp. The clinicopathologic findings and demonstration of the amebic organisms using immunohistochemical techniques, culture, polymerase chain reactions, and electron microscopy are presented...
- Fatal granulomatous Acanthamoeba encephalitis mimicking a stroke, diagnosed by correlation of results of sequential magnetic resonance imaging, biopsy, in vitro culture, immunofluorescence analysis, and molecular analysisMehri S McKellar
Division of Infectious Diseases, Department of Internal Medicine, University of Utah, Salt Lake City 84132 2405, USA
J Clin Microbiol 44:4265-9. 2006..We report a unique case of granulomatous amebic encephalitis that was proven pathologically with progressive radiological findings on MRI...
- In vitro culture, serologic and molecular analysis of Acanthamoeba isolated from the liver of a keel-billed toucan (Ramphastos sulfuratus)Govinda S Visvesvara
Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
Vet Parasitol 143:74-8. 2007..Further, we characterized the cultured amoeba and also the amoeba in the liver tissue as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene...
- Length and sequence heterogeneity in the mitochondrial internal transcribed spacer of Acanthamoeba sppDolena R Ledee
Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida 33136, USA
J Eukaryot Microbiol 56:257-62. 2009..Also, the potential for an additional type of editing not described previously for Acanthamoeba, involving purine to pyrimidine transversions was observed...