Laboratory Research Interests:
Acanthamoeba
Acanthamoeba is a genus of small free-living amoebae that
are ubiquitous in nature. The organisms are opportunistic pathogens that
cause several rare diseases in humans. These include a central nervous
system disease known as Granulomatous Amoebic Encephalitis (GAE), a sight-threatening
eye disease known as Acanthamoeba keratitis (AK), and various secondary
infections associated with immunocompromised individuals such as AIDS patients.
Acanthamoebae also have been associated with disease in a variety of animals
other than humans.
Cell and molecular biology of growth
and differentiation.
Early studies focused on relationships between environmental factors and
cell growth, multiplication and cyst formation [Reviewed in Byers, 1979].
We studied the induction of encystment by glucose-acetate starvation [Byers,
Akins, Maynard et al., 1980], the uncoupling of nuclear and cytoplasmic
division by physical agitation [James & Byers, 1967], photoinhibbition
of cell multiplication [Dolphin,1968], changes during culture aging in
oxygen consumption and RNA, DNA, protein and polysaccharide levels [Byers,
Rudick & Rudick, 1969], relationships between nucleic acid synnthesis
and degradation during growth and encystment [V. Rudick, 1971; King, 1980],
evidence for lysosomal enzymes and changes in acid hydrolase activity during
growth and encystment [Martin & Byers, 1976, 1977], morphological changes
and requirements for macromolecule synthesis during excystment [Mattar
& Byers, 1971], and polyamine content and metabolism during growth
and differentiation [Kim, Sobota, Bitonti et al., 1987]. Collectively,
these studies revealed extensive changes in macromolecule content and metabolism
that begin while amoebae are still in mid-logarithmic growth, thus, bringing
into question the common practice of using late logarithimic phase cultures
for many biological studies.
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Induction of encystment by metabolic inhibitors and
the encystment enhancing activity EEA.
We used metabolic inhibitors in an attempt to understand the initiation
of cell differentiation (encystment). Agents tested included hydroxyurea
[King, 1975], mitomycin C [Kuhns, 1975], inhibitors of polamine metabolism
[Kim, Byers & McCann, 1987], and various inhibitors of mitochondrial
macromolecule synthesis [Akins & Byers, 1980; Akins, 1981]. One of
the most important result of these studies was the discovery of a potent
Encystment Enhancing Activity (EEA) that was released from amoebae during
encystment was discovered [Akins & Byers, 1980; Akins, 1981; Akins,
Gozs & Byers, 1985]. The activity was due to a small phosphorylated
molecule that has not yet been further identified. Due to the ready stimulation
of encystment by inhibitors of mitochondrial macromolecule synthesis and
the association of EEA with a mitochondrial fraction, it was speculated
that it might be a mitochondrial product [Akins, 1981]. A second
important result from the studies of drug-induced encystment was the recognition
that some of the agents commonly used to treat Acanthamoeba keratitis probably
have the counterproductive effect of inducing cyst formation.
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Antibiotic resistance.
In early studies on cell differentiation, cultures were consistently responsive
to metabolic inhibitors that induced encystment. Later, it became
evident that cultures were developing resistance to the inhibitors. Therefore,
we began a study of antibiotic resistance. We found that Acanthamoeba
readily developed resistance of cell multiplication to several antibiotics
that affected mitochondrial processes as well, certainly, as other targets
[Seilhamer & Byers, 1978; Byers, Akins & seilhamer, 1981; Akins,
1981]. It then was demonstrated that ATPase activity of mitochondria
isolated from oligomycin resistant amoebae also exhibited resistance to
the inhibitor [Seilhamer & Byers, 1982]. Several of the inhibitors
tested were related to diamidines used as chemotherapeutic agents for Acanthamoeba
keratitis. In a recent study, we were able to confirm that a single strain
of Acanthamoeba developed resistance to propamidine in the eye of a patient
during therapy for keratitis. Thus, we recommend the desirability of using
a dual drug or multidrug therapy for treatment of Acanthamoeba infections
[Ledee, Seal & Byers, In preparation].
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Mode of reproduction.
The question of whether Acanthamoeba reproduces entirely by asexual means,
or whether there is some mechanism for genetic exchange has been a continuing
puzzle. The availability of drug resistance markers enabled us to
explore whether they could be used to determine if genetic
exchange occurs in Acanthamoeba. Approaches used included co-incubating
amoebae with different resistance markers [Akins, 1981], or incubating
drug-sensitive amoebae with mitochondria from drug-resistant amoebae [D.
Zarley, unpublished]. Ambiguous results were obtained. In some cases,
recombination of resistance markers, or the acquisition of resistance following
exposure to resistant mitochondria, appeared to be achieved, but these
effects were short-lived and not consistently repeatable.
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Molecular phylogeny-based taxonomy.
Sequences
of nuclear and mitochondrial small subunit rRNA genes and mitochondrial
tRNA genes.
In the 1980's, ophthalmologists became increasingly interested in Acanthamoeba
keratitis. This generated concerns about the reliability of methods for
identification and classification of Acanthamoeba isolates. Identification
of the amoebae at the genus level is not difficult for individuals trained
in recognition of Acanthamoeba, but is a problem for the untrained. Classification
at the species level is a problem, however, even for experts. Better
criteria are needed for assessing the relatedness of various isolates both
for clinical purposes and for basic biological studies. Thus, we turned
our attention to solving this problem. Our first goal was to find
a reliable molecular basis for examining relaltionships among isolates
and creating a molecular phylogeny-based taxonomy of the genus. Our
second goal was to develop very sensitive probes that could detect and
reliably identify amoebae in infections. Similar probes should be
useful for environmental/biodiversity studies as well.
Restriction fragment length polymorphisms of
mitochondrial DNA (mt RFLP).
We first used mtRFLP to differentiate isolates of Acanthamoeba [Zarley,
1979; Bogler, Zarley, Burianek et al., 1983; Byers, Bogler & Burianek,
1983; Byers, 1983; Hugo, 1986]. We abandoned these studies because they
did not look promising. However, other laboratories subsequently
have used them sucessfully.
Multiple alleles.
In the large majority of isolates studied, we found evidence for only one
nuclear 18S rDNA allele. In a few cases, evidence was found for more
than one allele [Ledee, 1995; Ledee, Fuerst & Byers, in preparation].
No evidence was found for multiple alleles of the mitochondrial 16S rDNA.
Where multiple alleles occurred, they all fell within the same 18S rDNA
sequence type (i.e. lineage).
Group I introns of nuclear 18S rRNA genes.
Sequence types T3 and T5, which are the A. griffini and A. lenticulata
lineages, are unique in the genus because they each include some isolates
with group I introns in the 18S rRNA genes [Gast, Fuerst & Byers, 1994;
Schroeder-Diedrich, Fuerst & Byers, Submitted]. Individual isolates
have a maximum of one of these introns. The intron is found at a single
position In A. griffini, but at one of three different positions in A.
lenticulata. The intron sequences are very different at the three different
positions in A. lenticulata. Distributions of introns among isolates within
these two species suggest that all four intron types were acquired following
branching of the lineages and that intron loss also may have occurred several
times subsequently. The sequence of the A griffini intron suggests that
it may have been acquired horizontally from a green alga, possibly a prey
organism, but there is no strong evidence that the same is true for any
of the A. lenticulata intron types.
Mitochondrial tRNA sequences and editing.
Evidence for mitochondrial tRNA editing in Acanthamoeba originally was
demonstrated in the laboratory of Michael Gray. Our analysis of sequences
for 5 tRNA genes from each of 16 isolates supports and supplements the
conclusions from Gray's laboratory. The observed sequence variation also
is consistent with the 18S rRNA gene phylogeny [Ledee, Fuerst & Byers,
In preparation].
Sequences of nuclear and mitochondrial small subunit
rRNA genes and mitochondrial tRNA genes.
We next turned to sequences of nuclear small subunit 18S rRNA genes (18S
rDNA) for phylogenetic studies because of the increased information content
and the large amount of available information about rRNA phylogeny in other
organisms. We now have partial to complete 18S rDNA sequences for at least
75 isolates. To date, this approach has produced a phylogeny for
17 of the 20+ described species of Acanthamoeba and has identified 12 distinct
rDNA lineages referred to as sequence types T1-T12 [Gast, Fuerst &
Byers, 1966; Stothard et al., 1998]. Support for the 18S rDNA
phylogeny has been obtained from examination of the mitochondrial 16S rRNA
gene phylogeny and sequence variation among mitochondrial tRNA genes [Ledee,
1995; Ledee, Fuerst & Byers, in preparation]. Preliminary data
suggest that there is some consistency between the mitochondrial 16S rRNA
gene sequence data and RFLPs for the complete mitochondrial genome.
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Use of 18S rDNA sequences in identification of isolates.
Direct sequencing.
Practical application of the identification methods is currently underway.
A comparison of 18S rDNA sequences and the presence of a unique intron
allowed us to identify A. griffini from an AK isolate and to establish
a connection between the infectious agent, the patient's contact lens case,
and the home water supply [Ledee. Hay. Seal et al., 1996]. Numerous
other human and environmental isolates also have been identified by sequencing.
In most cases, identification was based on the complete 2300-3000 bp gene
sequence, but a much shorter "diagnostic fragment" now is in use.
Phylogeny-based PCR and in situ probes.
The 18S rRNA gene sequences have been used as a basis for designing PCR
and FISH probes that are either genus-specific or specific for the T4 sequence
type. [Stothard, Seal & Byers, in preparation; Schroeder-Diedrich,
Seal & Byers, in preparation] Both types of probes have been tested
successfully on laboratory cultures of all 12 sequence types, on human
corneal scrape specimens and on acanthamoebae isolated from fish tissues.
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