International Seminar on Amoebiasis

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International S e m i n a r on Amoebiasis W.A. Petri, Jr The I lth Seminar on Amoebiasis was held in Mexico City from 15 to 17 November 1989. Over 100 scientists from 14 countries participated in the meeting, which was organized by Drs Librado Ortiz and Norberto Trevino, Directors of the Center for the Study of Amoebiasis, Mexico City, and Louis Diamond of the National Institutes of Health, USA. Rapid and exciting developments in diverse areas of Entamoeba histolytica research make it possible to cover only some of the many interesting topics discussed. P a t h o g e n i c vs N o n - p a t h o g e n i c Amoebae

Over 3000 clinical isolates ofE. histolytica have been grouped into pathogenic or non-pathogenic zymodemes (PZs and NPZs, respectively) on the basis of isoenzyme analysis. Sharon Reed (University of California, San Diego, USA) reported that there has not been a single NPZ identified from a patient with invasive amoebiasis and only 25 PZs isolated from asymptomatic individuals.The controversy over whether PZs and NPZs represent distinct species or interconvertable phenotypes has not been completely resolved. While most investigators have not seen zymodeme switching in culture, David Mirelman (Weizman Institute of Science, Israel) published work in 1986 showing that a cloned NPZ isolate cultured with 7-irradiated bacteria switched to PZ after 21 days. Mirelman communicated the recent results of Brian Andrews (University of Bergen, Norway) who has apparently also observed zymodeme switching on co-culturing a cloned isolate with Crithidia. These results are hard to reconcile with other data from Mirelman's group that show that PZs and NPZs can be distinguished by DNA probes to repetitive ribosomal RNA genes, and that amino acid substitutions exist in the ferredoxin genes of PZs and NPZs. Egbert Tannich (Bernhard Nocht Institute, FRG) described two distinct Southern blot patterns for PZ and NPZ genomic DNA probed with a cDNA for a PZ membrane protein (cEhPI) as well as the PZ actin and PZ cysteine proteinase genes. After low stringency hybridization these PZ probes on Southern blots showed there were two different RFLPs for PZs and NPZs, but

no hybridization of the PZ probes to NPZ genomic DNA was seen at high stringency. Differential gene amplification between PZs and NPZs has been proposed to explain the ability of ribosomal DNA probes to distinguish PZs from NPZs, but even PCR amplification could not detect a PZ cEhPI gene in NPZ amoebae. The NPZ equivalent of the cEhPI gene shared only 88% homology with the PZ gene. With mounting evidence that PZs and NPZs are genetically distinct, a major topic of discussionwas the production of monoclonal antibodies (mAbs) and DNA-based tests to distinguish them. Tannish, Mirelman and John Samuelson (Harvard School of Public Health, USA) independently presented results from assays, using DNA probes to ribosomal DNA and a membrane protein gene, that identified either directly, or by using polymerase chain reaction (PCR) amplification, less than 100 PZ or NPZ trophozoites. The detection and differentiation of PZs from NPZs was also described by John Ackers (London School of Hygiene and Tropical Medicine, UK) with a mAb to a currently uncharacterized antigen and by our group, with a mAb to the galactose lectin. Monoclonal antibody detection appeared to be as sensitive as the DNA probes. Since NPZs are almost ten times more common than PZs, the availability of mAb or DNA tests promises to eliminate the potentially unnecessary treatment of individuals colonized with NPZs. Adherence

Proteins

Adherence is important not only for the colonization of the large bowel but

also for the contact-dependent cytolysis of the host cells from which E. histolytica takes its name. The complexity of the adherence process is reflected in the increasingnumber of proteins implicated (Table I). The 260 kDa galactosebinding lectin mediates adherence to human colonic mucin glycoproteins, suggesting that it is involved in the initial colonization and invasion of the colon. We reported the effects of mAb binding to six non-overlapping epitopes on the 170 kDa subunit of the lectin on adherence to Chinese hamster ovary (CHO) cells and colonic mucins. Surprisingly, mAb to epitopes I and 2 increased the galactose-binding activity of the lectin up to fivefold and, furthermore, the binding activity of purified lectin to CHO cells also increased. The direct activation of adherence by anti-lectin mAb suggested that adherence can be dynamically regulated via changes in lectin activity. Antibody-mediated enhancement of adherence may represent a novel means of immune evasion by the parasite. A role for the galactose lectin in signalling amoebic actin polymerization was suggested by Gordon Bailey (Morehouse School of Medicine, Atlanta, USA). Intracellular polymerization of actin occurs as amoebae adhere to and ingest either red blood cells or liposomes composed of red blood cell lipids. Bailey showed that while many different kinds of liposomes could bind to amoebae, only synthetic liposomes containing galactose-terminal glycosphingolipids or glycoproteins stimulated actin polymerization. Isaura Meza (CINVESTAV, Mexico) reported the cloning of the 220 kDa chitotriose lectin. Southern blots indicated that it is a single copy gene and that

Table I. Adherence proteins ofEntamoeba histolytica Molecular mass

Recognizes:

Mediates adherence to:

260 kDa heterodimer (I 70 and 35 kDa subunits)

Galactose, fibronectin

Human colonic mucins, Chinese hamster ovary cells

I

220 kDa

Chitotriose, hyaluronate

Erythrocytes, M D C K cells

2

I 12 kDa

Unknown

Erythrocytes

3

Fibronectin, laminin

Extracellular matrix

4

37 kDa a

Ref.

The 37 kDa fibronectin receptor appearsto be identicalto the 35 kDa light subunitof the galactose lectin. ~) 1990,ElsevierSciencePublishersLtd, (UK) 0169-4707/90/$02.00

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it is also present in E. invadens and the Laredo strain of Entamoeba. Meza showed that the mAb against the chitotriose lectin halved the level of erythrocyte adherence and phagocytosis. The 37 kDa fibronectin receptor has also been found by Meza's group to mediate binding to laminin, another constituent of the extracellular matrix. During the discussion of Meza's paper, Barbara Mann (University of Virginia, USA) commented on experiments measuring the binding of [~251]-Iabelled fibronectin to the purified galactose lectin, which indicate that the 37 kDa fibronectin receptor and the 35 kDa lectin subunit are one and the same. Esther Orozco (CINVESTAV, Mexico) gave an update on the 112 kDa amoebic protein identified on the western blots by mAbs that inhibit erythrocyte adherence and phagocytosis by 50%. Purification of the protein has proved difficult, bu~ phagocytosisdeficient mutants produced by Orozco may yield new insights into its function.

Fig. I. Entamoeba histolytica trophozoites

(arrowheads) adhering to mucins in rat colon. Note the goblet cell with apical protrusion (arrow) in the interglandular epithelium. Scale bar is 0.06 rnrn. (Hernatoxylin-eosin, ×312.)

Resistance to Infection Human colonic mucin glycoproteins inhibit the adherence and lysis of target cells in vitro by binding to the 260 kDa galactose lectin. Proteolytically degraded mucins were reported b), Kris Chadee (McGill University, Canada) to be of equal or even greater potency than native mucin (Fig. I ). It was not unexpected that proteolytically degraded mucins would be equally active as it is the galactose-terminal carbohydrate portion of the mucin that has be.en shown to inhibit adherence. However, the observed increase in inhibition of adherence raises the interesting possibility that there may be an endogenous galactosebinding lectin present in native mucins, which is removed by protease treatment. Anti-amoebic cell-mediated immunity (CMI) has been associatec with protection from liver abscesses in a gerbil model of the disease. Jonathan Ravdin (University of Virginia, USA) reported that only gerbils immun'zed with an insoluble form of galactose lectin in Freund's adjuvant developed an antilectin CMI response and were protected from liver abscesses. Immunization with a soluble preparation of the lectin triggered humoral but not CMI responses, and the animals were not protected from subsequent intrahepatic challenge with E. histolytica. Genetic susceptibility to invasive amoebiasis in humans has been investigated by Roberto Kretschmer's group

(Centro Medico Nacional, Mexico). They have found that the HLA DR3 allele was present in 36% of I I 0 patients with liver abscesses and 13% of I I0 uninfected individuals. Such advances in understanding host susceptibility combined with recent advances in determining genetic differences in PZs and NPZs have begun to explain why only a minority of patients infected with E. histolytica develop the disease.

Proteinases The cysteine proteinase was reported to have been cloned by both Jacques Bouvier (University of California, San Francisco, USA) and Tannich. It has a similar amino acid sequence to eukaryotic cathepsin B- or C-like enzymes. Northern and Southern blot analysis showed the proteinase gene to be present in both PZ and NPZ amoebae. Bouvier mentioned that the purified proteinase would release cultured cells from a monolayer without necessarily killing the cells. Reed presented data that showed that the proteinase is responsible for the activation of the alternative complement pathway by amoebae.

of the human P-glycoprotein using PCR in E. histolytica. The P-glycoprotein is a pump that removes drugs from cells; its enhanced expression appears to be responsible for multi-drug resistance in mammalian cells and Plasmodium. Northern blots of amoebic RNA with the PCR products identified a 4.5-5.0 kb mRNA, the prevalence of which was increased up to sevenfold in emetineresistant mutants of E. histolytica but it was not entirely clear whether gene amplification occurred, in addition to increased transcription. The 12th Seminar on Amoebiasis will be held in November 1991 in Mexico City and will be named in honour of the late Dr Bernardo Sepulveda, a pioneer in the study of the immunology of amoebiasis. The combination of molecular, biochemical and immunological approaches currently being used to examine this parasite should lead to significant advances in the next two years, with the promise of another lively meeting. References I Petri, W.A., Jr et al. (1989)J. Biol. Chem. 264, 3007-3012 2 Meza, I. et al. ( 1987)J. Infect. Dis. 156,798-805 3 Arroyo, R. and Orozco, E. (1987)/Viol. Biochem. Parasitol. 23, 151 - 158 4 Talamas-Rohana, P. and Meza, I. (1988)J. Cell Biol. 106, 1787-1794

Bill Petri is at the Departments of Medicine and Microbiology, University of Virginia, Charlottesville, VA 22908, USA.

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Drug Resistance Drug resistance in amoebae has fortunately rarely been encountered. Samuelson has found conserved regions

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