netic relationship between patliogenic and non- 
pathogenic E. histolytica strains. 
The 112-kDa Adhesin of Entamoeba histolytica 
Adherence, phagocytosis, and secretion are three 
key events in target cell destruction by E. histoly- 
tica. The exceptionally phagocytic and vesicle-rich 
trophozoites, the infective phase of this parasite, 
constitute an excellent model for studying these 
functions. Drs. Mario Alberto Rodriguez and Rossana 
Arroyo, in Dr. Orozco's group, identified and char- 
acterized E. histolytica's 1 1 2-kDa adhesin, which is 
involved in phagocytosis and cytopathogenicity and 
plays an important role in the virulence of the para- 
site. It is absent or altered in nonpathogenic tropho- 
zoites and in virulence-deficient mutants. Recent 
experiments carried out by Dr. Christine Rigotier 
and Guillermina Garcia-Rivera showed that the ad- 
hesin is also involved in traffic and compartmental- 
ization through vesicular intermediates en route to 
the plasma membrane to be secreted. Such an adhe- 
sin was not found in virulence-deficient mutants. 
The 1 1 2-kDa adhesin has been partially purified 
by immunoaffinity chromatography using a mono- 
clonal antibody. Two proteins of 70 and 55 kDa 
were also released from the immunoaffinity col- 
umn. The three proteins were recognized by mono- 
specific polyclonal antibodies against the 112-kDa 
adhesin. The adhesin, purified from preparative 
polyacrylamide gels, gave the same 70- and 55-kDa 
proteins when incubated in di-dithiothreitol. 
Proteins of 11 2 and 70 kDa presented protease 
activity that was detected by their ability to degrade 
gelatin. A hypothesis is that the 1 1 2-kDa protein was 
broken down into these two peptides. Experiments 
are being performed to learn whether this mecha- 
nism occurs in vivo and whether it is related to the 
cytopathogenicity of the parasite. 
Entamoeba histolytica Presents Variable 
Virulence, Selectively Virulent 
Antigens, and Genome Rearrangements 
through Axenization 
Mechanisms involved in the expression of patho- 
genicity of E. histolytica were investigated through 
the process of axenization of a cloned nonpatho- 
genic isolate, MAV-CINVESTAV, obtained from an 
asymptomatic carrier. Phenotypic characteristics 
traditionally related to the pathogenicity of this para- 
site, such as zymodeme, virulence, antigenic ex- 
pression, presence of the 11 2-kDa adhesin and pro- 
teases, as well as the amount of DNA per cell, 
molecular karyotype, and organization of some 
virulence-involved genes, varied remarkably. 
Cloned strain MAV-CINVESTAV (clone MAV-1 ), cul- 
tured under monoxenic and polyxenic conditions 
(MAVmx and MAVpx, respectively), presented non- 
pathogenic zymodeme and did not express viru- 
lence. However, trophozoites cultured under 
axenic conditions (MAVax) showed pathogenic zy- 
modeme, a high rate of phagocytosis, and ability to 
damage target cells. 
Virulence-involved antigens, such as the 1 1 2-kDa 
adhesin and the major cysteine protease, were ex- 
pressed only in MAVax trophozoites. Striking 
changes in molecular karyotype, genome rearrange- 
ments, and gene amplification of virulence-related 
genes were also exhibited by the trophozoites 
through the axenization process in correlation with 
the expression of virulence in vitro. These results 
suggest that a cloned population of E. histolytica is 
able to modulate the expression of its virulence, 
probably through genomic rearrangements, includ- 
ing gene amplification, with the influence of me- 
dium conditions. 
Genomic Variability in Closely 
Related Clones of Entamoeba histolytica 
The molecular basis of the variability of E. histo- 
lytica was studied in clones A and L6, both obtained 
from the heterogeneous strain HMl-IMSS and, in 
clone C2, derived from clone A. By differential 
plaque hybridization of clones A and L6, the group 
isolated a 3.5-kbp cDNA clone, pMD. Certain pMD 
fragments recognized several transcripts in the three 
clones. However, another pMD fragment hybridized 
with a transcript of 0.63 kb was found only in clone 
A trophozoites. Southern blot analysis of both chro- 
mosomes and digested total DNA showed that all 
pMD fragments are encoded by a linked piece of 
DNA located in the 1.3- and 1.4-Mb chromosomes 
and in a 6-kbp EcoRl-EcoRl DNA fragment. 
Polymerase chain reaction (PCR) experiments 
demonstrated that the 0.63-kb differential tran- 
script is absent or highly modified in the DNA of 
clones L6 and C2 trophozoites. The genetic rela- 
tionship between the clones was verified by DNA 
polymorphism of several genes. Enzymatically di- 
gested DNA from clones A and C2 yielded almost 
identical restriction patterns, while differences be- 
tween them and clone L6 were evident. 
However, trophozoites of clone A cultured in 
various laboratories for several years gave different 
polymorphic fragments for many genes. MAV tro- 
phozoites did not hybridize with the differential 
pMD fragment, indicating that this sequence is ab- 
sent or highly modified in them. These results indi- 
cated that E. histolytica presents a high genomic 
INTERNATIONAL RESEARCH SCHOLARS 523 
