Cellular and Molecular Basis of Variability 
in Entamoeba histolytica 
Esther Orozco, Ph.D. — International Research Scholar 
Dr. Orozco is Professor of Genetics and Molecular Biology in the Department of Experimental Pathology at 
the Center for Research and Advanced Studies, National Polytechnic Institute, Mexico City. She received 
her bachelor's degree in chemistry and biology from the University of Chihuahua, Mexico, and her Ph.D. 
degree from the National Polytechnic Institute. She has been a visiting professor in several institutes 
around the world, including Harvard School of Public Health in Boston and the Weizmann Institute of 
Science in Israel. Dr. Orozco counts among her honors a Guggenheim Foundation Award and the 
Dr. J. Rosenkranz 1991 Award given by Syntex. 
MORE than 500 million people throughout 
the world are infected by Entamoeba his- 
tolytica, the protozoan parasite responsible for 
human amebiasis. Ninety percent of those in- 
fected do not present clinical symptoms, while 
the rest develop colitis, dysentery, or hepatic 
abscesses. 
In 1925 Brumpt proposed the possible exis- 
tence of two E. histolytica species, one patho- 
genic (P) and the other nonpathogenic (NP), and 
he and others have continued to advance this pos- 
sibility. There are data both supporting and op- 
posing this concept. For instance, migration pat- 
terns of several enzymes in a high number of 
amebic isolates has led to the establishment of 
more than 20 E. histolytica zymodemes. Interest- 
ingly, the zymodemes of P and NP trophozoites 
differ. 
According to the opinion of several groups, zy- 
modemes and virulence are stable phenotypes. 
However, experimental evidence against the sta- 
bility of zymodemes and virulence has also been 
documented. Resolution of this controversy is of 
fundamental importance clinically. If E. histoly- 
tica comprises two species, cyst passers spread- 
ing the NP form are not a public health menace, 
and asymptomatic carriers need not be treated. 
However, if the harmless E. histolytica can turn 
into a virulent one, a different decision may be 
indicated. In any case, the molecular basis of con- 
version, if it occurs, would be a highly interesting 
phenomenon to study. 
We have focused this controversy by 1 ) investi- 
gating cloned E. histolytica populations after 
their isolation from asymptomatic carriers through- 
out the axenization process, 2) comparing E. his- 
tolytica clones with diverse origins, and 3) 
detecting and cloning DNA sequences present 
only in E. histolytica clones that express 
pathogenicity. 
Study of NP E. histolytica Throughout 
Axenization 
Cloned trophozoites (MAV-1), with an initial 
NP zymodeme I and isolated from an asymptom- 
atic carrier, were cultured under polyxenic, 
monoxenic, and axenic conditions. Zymodeme I 
of polyxenic MAV-1 trophozoites, cultured in 
Robinson's medium, presented an NP zymodeme 
XII when trophozoites were cultured in Jones' 
medium. Monoxenic trophozoites grown in the 
presence of Fusobacterium symbiosum re- 
tained NP zymodeme I (with slow-running hexo- 
kinases [HKs]), an a-phosphoglucomutase (PGM) 
band, and lack of pathogenicity. Surprisingly, 
when MAV-1 trophozoites were grown under 
axenic conditions, the original NP zymodeme 
switched to a P zymodeme, with fast-running HKs 
and a /3 PGM. Virulence, defined as the tropho- 
zoites' ability to infect experimental animals and 
damage target cells, was also expressed. 
The analysis of E. histolytica zymodemes indi- 
cates that P as well as NP trophozoites have a set 
of genes for a given enzyme. Excluding the fast- 
running HKs and the /3 PGM that have not been 
reported for NP isolates, all other isoenzymes are 
expressed in P and NP trophozoites, including 
slow-running HKs. A certain type of bacteria in 
the medium could be participating in the regula- 
tion of isoenzyme expression. We will continue 
our studies on the factors involved in the expres- 
sion of isoenzymes in P and NP trophozoites, in 
order to look for conclusive evidence on zymo- 
deme and virulence switching — evidence that 
should dissipate skepticism about conversion. 
Differences in Genetically Related Clones 
Functional and biochemical dissimilarities 
mark clones of diverse origin. P trophozoites 
show a high rate of phagocytosis, destroy cell cul- 
ture monolayers, and produce hepatic abscesses 
in experimental animals. On the contrary, NP tro- 
phozoites generally do not show these virulence- 
related properties. Fortuitously, they express low 
virulence. 
Monoclonal and polyclonal antibodies against 
certain antigens can distinguish between P and 
NP trophozoites. The presence of the major ame- 
bic cysteine protease involved in the cytopathic 
effect of trophozoites is also specific to P tropho- 
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